HORMONE-INDUCED SIGNALING DURING MOSS DEVELOPMENT.

Understanding how a cell responds to hormonal signals with a new program of cellular differentiation and organization is an important focus of research in developmental biology. In Funaria hygrometrica and Physcomitrella patens, two related species of moss, cytokinin induces the development of a bud during the transition from filamentous to meristematic growth. Within hours of cytokinin perception, a single-celled initial responds with changes in patterns of cell expansion, elongation, and division to begin the process of bud assembly. Bud assembly in moss provides an excellent model for the study of hormone-induced organogenesis because it is a relatively simple, well-defined process. Since buds form in a nonrandom pattern on cells that are not embedded in other tissues, it is possible to predict which cells will respond and where the ensuing changes will take place. In addition, bud assembly is amenable to biochemical, cellular, and molecular biological analyses. This review examines our current understanding of cytokinin-induced bud assembly and the potential underlying mechanisms, reviews the state of genetic analyses in moss, and sets goals for future research with this organism.

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title: Programmed Changes in Form during Moss Development.
abstract: Picture an organism that begins development as a germinated spore to give rise to a filament via tip growth. Within days of germination, and with the involvement of only a few cells and cell types, the pattern of growth changes dramatically from two dimensional to three dimensional as a leafy structure is produced on the filament. With this simple picture in mind, we can begin to think about vegetative development in moss. In this review, we introduce the reader to the processes involved in moss development, speculate on some of the underlying mechanisms, outline some of the advantages of using moss development to understand elements of general eukaryotic development, and identify areas that require additional research and clarification. We focus our discussion on the development of Funaria hygrometrica grown in culture, with additional information from studies using Physcomitrella patens. The very early stages of moss development are characterized by cellular differentiation during filament growth. Spore germination leads to the formation of a filament that is made up of a tip cell (an apical cell) and a linear array of subapical cells that are produced by successive divisions of the tip cell. These subapical cells have walls that are perpendicular to the filament axis and filled with large round chloroplasts, hence their name, chloronema (Figure 1A). As is characteristic of tip-growing organisms, the subapical chloronema cells, which are -1 O0 pm in length, do not grow. The tip cell continues to elongate, reaches a maximum length of -250 pm, and divides every 1 O to 12 hr to extend the filament. Chloronema filament growth continues until, in response to increases in light and auxin, the appearance of the chloronema tip cell begins to change, ultimately giving rise to the second filament cell type, the caulonema. Fully developed caulonema tip cells differ from chloronema tip cells in severa1 important respects. They grow much longer (up to 400 pm), divide more often (every 5 to 6 hr), and possess fewer, smaller, and more elliptical chloroplasts. During the transition from chloronema to caulonema, the newly formed filament cells appear transitional, but after 5 to 6 days, the caulonema cells are long (-250 pm), nearly clear, and have cross walls that are oblique to the filament axis (Figure 1 B). Once caulonema cell formation has begun, a new axis of developmental polarity is set up as an initial cell is formed.

title: Growth regulation and development in Physcomitrella patens: an insight into growth regulation and development of bryophytes
abstract: Observations made on the effects of auxin, of cytokinin and of various light treatments on cultures of wild-type Physcomitrella patens, grown either on solid medium or in continuously-replaced liquid medium, have allowed us to suggest probable roles for these factors during the early stages of normal gametophyte development. Further data obtained by studying mutants affected in their responses to one or both types of hormones or to light have supported our model of gametophyte development and enabled us to elaborate it.

title: MORPHOGENESE DER LAUBMOOSE
abstract: INHALT I.  Sporenkeimung und Grundlagen der Protonemadifferenzierung . . . . . 111. Differenzierung des Laubmoosprotonemas , . . . . . . . . ( I ) Die Differenzierungsformen . . . . . . . . . . (2) Bedingungen fur die Differenzierung . . . . . . . . . (3) Spiralwachstum der Protonemen . . . . . . . . . . IV. Die Determinierung der Stiimmchenscheitelzelle . . . . . . . . ( I ) Der Ort der Moosknospenanlage . . . , . . . . . . (2) Bedingungen fur die Knospenbildung . . . . . . . . (3) Die Bedeutung von Wuchsstoff und ahnlichen Substanzen fur die Knospenbildung V. Entwicklungund Differenzierung der St-mchen . . . . . . .

title: Studies on cytokinin-controlled bud formation in moss protonemata.
abstract: Application of cytokinins to moss protonemata of the proper physiological age causes bud formation on specific cells (caulonema). During the early stages of their development, buds revert to protonemal filaments if the cytokinin has been removed by washing the protonemata. This indicates that the hormone is not acting as a "trigger" but has to be present during a critical period of time until differentiation is stabilized. Autoradiographs of protonemata treated with a labeled cytokinin, benzyladenine-benzyl-7-(14)C, show a striking accumulation of the radioactivity in caulonema cells which are in the stage of bud formation, and in the buds themselves. Cells which did not react to the hormone contained very little radioactivity. The accumulation of benzyladenine in the "target cells" may be due to the presence of binding sites which, in turn, may distinguish responding cells from non-responding ones.

title: The hormonal regulation of protonema development in mosses. II: The first steps of cytokinin action
abstract: Bud formation, a fundamental step in the morphogenesis of the protonema of Funaria hygrometrica and other mosses, is, as is well known, regulated by external cytokinins like kinetin, zeatin etc. The first visible effect of kinetin (10 μM l −1 ) is an arrest in the lengthwise growth of the side branches of caulonema. This effect is observed in less than three hours after hormone application. Thereafter, growth in the cell tip takes place in the widthwise dimension. Branches longer than 80 μm continue the filamentous growth. Within a few hours after removal of kinetin, tip growth is restored. From these observations it can be concluded that the effect of cytokinins is to change the growth form in the tip region of the side branches. Furthermore, the extent of this reaction depends on the caulonema cells themselves. For example, in isolated single cells it is always observed that the sixth cell of a filament produces the highest number of buds upon treatment with cytokinin.

title: Bud formation inFunaria: Organelle redistribution following cytokinin treatment
abstract: SummaryCytokinin stimulates caulonemata ofFunaria to undergo an asymmetric division leading to the gametophore. The earliest detectable event is a small protuberance at the distal portion of the cell accompanied by the reorganization of the underlying organelles into a polarized distribution reminiscent of a tip growing cell. Dictyosomes and associated vesicles accumulate in the protuberance directly beneath the plasma membrane with mitochondria subjacent to the vesicular layer. Endoplasmic reticulum lies beneath the mitochondrial zone directly above the large central vacuole, while chloroplasts are outside the bud. As development continues the bud elongates causing the outer cell wall to exfoliate. During the above events the nucleus migrates toward the bud site concomitant with an increase in the number of microtubules between the nucleus and the base of the outgrowth. Nucleoli, extruded from the nucleus during a previous division, persist as diffuse fragments within the protuberance. Upon reaching the bud site, division occurs with the developing phragmoplast being initiated distal to the caulonema tip cell. The former polarized distribution of the cytoplasm is altered as mitochondria, chloroplasts and small vacuoles become evenly dispersed throughout the cytoplasm; dicytosomes and endoplasmic reticulum occupy a cortical position. These events indicate a change from 2-D tip growth to 3-D diffuse growth. To quantify the ultrastructural changes associated with bud formation we performed a morphometric analysis of cells in various stages of budding. The relative volumes of dictyosomes and vesicles adjacent to the bud apex decrease during bud development coincident with an increase in these organelles in lower portions of the cytoplasm. Mitochondria and chloroplasts follow this same pattern although their highest relative volumes initially are 4 μm from the bud apex and outside the bud site, respectively. These data, as well as density profile topographic maps for vesicle fractions, support the contention that cytokinin induces a change in morphological symmetry and polarity in the fine structure ofFunaria.

title: Pre-prophase band of microtubules, absent from tip-growing moss filaments, arises in leafy shoots during transition to intercalary growth
abstract: In the presence of cytokinin, undetermined side branch initials of the moss, Physcomitrella patens, arc induced to form buds and then leafy shoots rather than to develop as tip-growing filaments. This represents a transition between the two modes of plant cell expansion–tip growth and uniform intercalary growth. The organization of microtubules in filaments is different from that in leafy shoots and can be traced back to the influence of phytohormones on side branch initials. Microtubules either focus at a particular region (as in tip-growing cells) or in the presence of high levels of cytokinin form swollen bud initials in which microtubules are more diffusely organized. Higher levels of cytokinin are capable of destabilizing tip microtubules in caulonemal filaments. Although caulonemata are not normally target cells, this implies that cytokinin may exert its morphogenetic effects by altering microtubule organization. 
 
 
 
In tip-growing filaments, interphase microtubules trace a meandering course through the cytoplasm towards the tip and are not for the main part associated with the plasma membrane as are cortical arrays. There is no pre-prophase band of microtubules to indicate the future division plane, even though the oblique division plane is known to be precisely controlled relative to environmental factors. This microtubule cycle contrasts with cells of the leafy shoots that develop from buds: in these, the interphase array is cortical, consisting of flat-pitched microtubular helices that do not focus upon a growing tip. It is now shown that pre-prophase hands occur at this stage. 
 
 
 
The absence of bands does not readily correlate with imprecise control of the division plane. Instead, it is proposed that the ability to form pre-prophase bands depends upon the arrangement of microtubules in the preceding interphase array. Ways in which bands might be formed are discussed and the generality of these ideas is tested by observations on higher plant cells.

title: To shape a cell: an inquiry into the causes of morphogenesis of microorganisms.
abstract: We recognize organisms first and foremost by their forms, but how they grow and shape themselves still largely passes understanding. The objective of this article is to survey what has been learned of morphogenesis of walled eucaryotic microorganisms as a set of problems in cellular heredity, biochemistry, physiology, and organization. Despite the diversity of microbial forms and habits, some common principles can be discerned. (i) That the form of each organism represents the expression of a genetic program is almost universally taken for granted. However, reflection on the findings with morphologically aberrant mutants suggests that the metaphor of a genetic program is misleading. Cellular form is generated by a web of interacting chemical and physical processes, whose every strand is woven of multiple gene products. The relationship between genes and form is indirect and cumulative; therefore, morphogenesis must be addressed as a problem not of molecular genetics but of cellular physiology. (ii) The shape of walled cells is determined by the manner in which the wall is laid down during growth and development. Turgor pressure commonly, perhaps always, supplies the driving force for surface enlargement. Cells yield to this scalar force by localized, controlled wall synthesis; their forms represent variations on the theme of local compliance with global force. (iii) Growth and division in bacteria display most immediately the interplay of hydrostatic pressure, localized wall synthesis, and structural constraints. Koch's surface stress theory provides a comprehensive and quantitative framework for understanding bacterial shapes. (iv) In the larger and more versatile eucaryotic cells, expansion is mediated by the secretion of vesicles. Secretion and ancillary processes, such as cytoplasmic transport, are spatially organized on the micrometer scale. The diversity of vectorial physiology and of the forms it generates is illustrated by examples: apical growth of fungal hyphae, bud formation in yeasts, germination of fucoid zygotes, and development of cells of Nitella, Closterium, and other unicellular algae. (v) Unicellular organisms, no less than embryos, have a remarkable capacity to impose spatial order upon themselves with or without the help of directional cues. Self-organization is reviewed here from two perspectives: the theoretical exploration of morphogens, gradients, and fields, and experimental study of polarization in Fucus cells, extension of hyphal tips, and pattern formation in ciliates. Here is the heart of the matter, yet self-organization remains nearly as mysterious as it was a century ago, a subject in search of a paradigm.

title: Visualization of the endoplasmic reticulum in living buds and branches of the moss Funaria hygrometrica by confocal laser scanning microscopy
abstract: Caulonemata of the moss Funaria hygrometrica were vitally stained with the fluorescent, lipophilic carbocyanine dye DiOC6(3) and examined via confocal laser scanning microscopy. Although DiOC6(3) stained nearly all of the organelles, cortical endoplasmic reticulum (ER) could be resolved under favorable conditions and appeared as a network of irregular polygons, interspersed with lamellar cisternae in some cell types. The pattern of cortical ER was examined first during side initial formation and then in young branches and buds. The ER network extends into the outgrowth of a developing side initial, keeping pace with elongation of the outgrowth. Prior to the cell division that cuts off the outgrowth from the underlying cell, the network in the outgrowth becomes tighter, i.e. the polygons become smaller. If the side initial develops as a branch, this somewhat tighter ER network is maintained in the tip-growing side branch. If the side initial develops as a bud, dramatic changes in both the configuration and the quantity of the ER network occur. Coincident with the apical swelling that marks the first visible sign of bud formation, the network becomes increasingly tighter until eventually the polygonal configuration is barely discernible. The increased coverage of the bud cortex by the ER network demonstrates that a significant increase in the quantity of membranes also takes place during bud formation in Funaria.

title: Cortical ultrastructure of freeze-substituted protonemata of the mossFunaria hygrometrica
abstract: SummaryThe ultrastructural organization of the cortical cytoplasm has been examined in caulonemata, branches and buds of the mossFunaria hygrometrica, which were prepared by rapid freeze-fixation and freeze-substitution (FS). The same structural components occur in the cortex of all three cell types: microtubules (MTs), endoplasmic reticulum (ER), coated and uncoated vesicles, coated pits, and dictyosomes. However, the configuration and density of the cortical ER varies between the three. Caulonemata have an open, polygonal network of ER associated with long MTs oriented mostly parallel to the length of the cell. Lamellar ER, covered with polysomes, is interspersed in the network. Branches have a more tightly arranged ER network, at places occurring in a thick layer, and occasional polysome-decorated lamellae. MTs, which extend to the tip of the branch, are oriented mainly parallel to the cell's long axis and are associated with the cortical ER. Buds have the tightest ER network, which is frequently arranged in a thick layer. Tubules in the polygonal ER of buds are densely covered with ribosomes, whereas tubules in the ER network of caulonemata and branches range from nearly smooth to moderately rough. Closely-spaced ER lamellae, with many polysomes, occur in some buds. The MTs of buds extend into the apical dome and are associated with the cortical ER, but are more randomly oriented than in caulonemata or branches. Close appositions between the ER and PM are observed in all three cells, but are more frequent in buds.

title: Cytokinin effect on branching and bud formation in Funaria
abstract: Cytokinins have two different effects on protonemata of Funaria hygrometrica. They induce branching of unbranched caulonemata and bud formation. Branching occurs after treatment with pico-molar concentrations of cytokinins whereas bud formation requires micro-molar concentrations. Both processes are therefore independently stimulated by cytokinins.

title: The isolation and physiological analysis of mutants of the moss, Physcomitrella patens, which over-produce gametophores
abstract: Several phenotypically distinct classes of gametophore overproducing mutants have been isolated in P. patens. Mutants belonging to one class resemble the wild-type strain grown on medium containing a high concentration (5–50μM) of exogenously supplied cytokinin. Mutants of this type can increase the production of gametophores in the wildtype strain by cross-feeding it through the culture medium. Mutants belonging to another class resemble the wild-type strain cultured on medium containing a lower concentration (50–500 nM) of exogenous cytokinin. Mutants of this kind cannot cross-feed the wild-type strain through the culture medium. A component, required by the wild-type strain for the initiation of gametophores in response to cytokinin, either is not formed or is not activated in the dark. Gametophore over-producing mutants may also be unable to synthesize/activate this component in the dark and thus, like the wild-type strain, they produce no gametophores in the dark.

title: GENETIC AND PHYSIOLOGICAL STUDIES OF THE EFFECT OF LIGHT ON THE DEVELOPMENT OF THE MOSS, PHYSCOMITRELLA PATENS
abstract: The germination of Physcomitrella patens spores only occurs when wet spores are exposed to light. Depending on their ripeness, spores require from 44 to 64 h illumination to bring about maximum germination. There is a lag period of about 15 h between the reception of sufficient light to elicit germination before germination can be observed. Wavelengths in the range 640–64080 nm are much more effective in inducing germination than longer or shorter wavelengths, but far‐red reversal of red light induction of germination has not been demonstrated. Light also has very marked effects on protonemal and gametophore development. In darkness, only caulonemata are produced, and these grow negatively geotropically. No new gametophores develop but existing gametophores grow negatively geotropically, etiolate and bear only scale leaves. In light, chloronemata, as well as caulonemata are produced, the former grow positively phototropically, while the latter grow at right angles to the direction of light, and neither cell type is sensitive to gravity. In the light, gametophores grow positively phototropically, are indifferent to gravity, produce large leaves and do not etiolate. All these responses to light by protonemata and gametophores are shown by cultures growing in a 23 h dark/l h red light cycle, but if this red light treatment is followed by 15min far‐red light, the effect of the red light is reversed, indicating an involvement of phytochrome in the mediation of these responses. Mutants showing abnormal growth in the dark have been isolated, as well as mutants having abnormal phototropic responses. The latter type has lost the phototropic response of both the protonemal cell types, as well as of gametophores, indicating that these different responses may share a common component.

title: Light dependency of the cytokinin-induced bud initiation in protonemata of the moss Funaria hygrometrica
abstract: Protonemata of the moss Funaria hygrometrica (Hedw.) are grown in large amounts in a liquid culture system, allowing repetitive dry weight measurements for growth control. Ten-day-old protonemata are submitted to various light conditions, in the presence of a cytokinin (iP), which is known for its activity in bud initiation in the moss protonema. The hormone has no influence on the growth rate. Growth and budding are shown to be tightly related. Light is necessary for the initiation of buds but not for their development (to some extent). Identical protonematal growth can be obtained in white, Ted or blue light, but not budding, which is reduced in red or blue light. The early morphological changes in red light, known as nutations, and the presumed inhibitor in blue light could be responsible for the reduced budding. Bud initiation in the dark on an organic medium is greatly enhanced if red light pulses are given at regular intervals. When the latter are followed immediately by far-red light pulses, the stimulating effect is partly suppressed, suggesting a phytochrome-controlled process. 
 
 
 
Experiments with DCMU showed a peculiar effect of this herbicide, which inhibited the use of exogenous sugar in the light but not in the dark.

title: LIGHT, TEMPERATURE AND CARBOHYDRATE REQUIREMENTS FOR SHOOT‐BUD INITIATION FROM PROTONEMATA IN THE MOSS PHYSCOMITRIUM TURBINATUM
abstract: A B S T R A C T Clonal protonemata of the moss Physcomitrium turbinatuin were grown on a mineral nutrient, sucrose, agar medium under a wide range of incandescent light intensity, temperature and photoperiod conditions. Shoot-bud differentiation was assayed as the amount of calendar time and total hours of illumination required between inoculation (protonemal inoculant) and initiationl (the one-celled stage) of the first bud in the colony. There was no correlation between bud initiation and amount of protonemal growth. When temperature was not limiting, bud initiation occurred upon giving a requisite light dose regardless of how the light was divided into photoperiods over various durations of calendar time. We interpret this "cumulative light-dose" effect to mean that budding was dependent upon a threshold level of some morphogenic substance which was synthesized in strict accordance to light dose and was held in store over dark periods. The relationship between time required for bud initiation and light intensity showed that the rate of synthesis of the substance was linear with the log of light intensity in the range from 30-715 ft-c. Experiments at low temperature (16 C) indicated that the light reaction was dependent upon substrates provided by dark reactions. Accumulation of the proposed substance was also dependent upon the sparing action of carbohydrate which could be provided either by optimal photosynthesis or exogenous addition of sugar. In the absence of added sugar time required for budding increased in direct relation to increasing proportions of dark petriods or decreasing light intensity below the optimal level.

title: Analysis of gametophytic development in the moss, Physcomitrella patens, using auxin and cytokinin resistant mutants
abstract: Mutants altered in their response to auxins and cytokinins have been isolated in the moss Physcomitrella patens either by screening clones from mutagenized spores for growth on high concentrations of cytokinin or auxin, in which case mutants showing altered sensitivities can be recognized 3–4 weeks later, or by non-selective isolation of morphologically abnormal mutants, some of which are found to have altered sensitivities. Most of the mutants obtained selectively are also morphologically abnormal. The mutants are heterogeneous in their responses to auxin and cytokinin, and the behaviour of some is consistent with their being unable to make auxin, while that of others may be due to their being unable to synthesize cytokinin. Physiological analysis of the mutants has shown that both endogenous auxin and cytokinin are likely to play important and interdependent roles in several steps of gametophytic development. Although their morphological abnormalities lead to sterility, genetic analysis of some of the mutants has been possible by polyethyleneglycol induced protoplast fusion.

title: Bud formation in Funaria hygrometrica: A comparison of the activities of three cytokinins with their ribosides
abstract: SummaryThe activities of the cytokinins benzyladenine (BA), 6-(3-methylbut-2-enylamino)purine (IPA) and zeatin in promoting bud formation in protonemata of Funaria hygrometrica (L.) Sibth. were compared to the biological activities of the corresponding ribosides. Cytokinins were found to have considerably higher hormonal activity as free bases than as ribosides. This observation will be discussed in relation to the chemical identity of the celldivision factor (CDF) isolated from crown gall tumors of Vinca rosea L.

title: Development of an efficient two-element transposon tagging system in Arabidopsis thaliana
abstract: SummaryModified Ac and Ds elements, in combination with dominant markers (to facilitate monitoring of excision, reinsertion and segregation of the elements) were introduced into Arabidopsis thaliana ecotype Landsberg erecta. The frequencies of somatic and germinal transactivation of the Ds elements were monitored using a streptomycin resistance assay. Transactivation was significantly higher from a stable Ac (sAc) carrying a 537 by deletion of the CpG-rich 5′ untranslated leader of the transposase mRNA than from a wild-type sAc. However, substitution of the central 1.77 kb of the transposase open reading frame (ORF) with a hygromycin resistance marker did not alter the excision frequency of a Ds element. β-Glucuronidase (GUS) or iaaH markers were linked to the transposase source to allow the identification of plants in which the transposase source had segregated away from the transposed Ds element, eliminating the possibility of somatic or germinal re-activation. Segregation of the excision marker, Ds and sAc was monitored in the progeny of plants showing germinal excision of Ds. 29% of the plants inheriting the excision marker carried a transposed Ds element.

title: Immunoassay and ultrastructural localization of isopentenyladenine and related cytokinins using monoclonal antibodies
abstract: Two hybridoma cell lines, J40-IV-A1 and J40-IV-C4 were obtained from a fusion of spleen cells of Balb/c mice immunized against an isopentenyladenosine-bovine serum albumin conjugate with X63. Ag 8.653 myeloma cells. These hybrids secrete monoclonal antibodies of the immunoglobulin G (IgG) class and share high affinities and specificities to isopentenyladenine and isopentenyladenosine suitable for the detection of femtomole amounts of these cytokinins in plant extracts by enzyme-linked immunosorbent assay (ELISA). One of the monoclonal antibodies (J40-IV-C4) has been employed to localize isopentenyladenine immunoreactivity in a cytokinin-over-producing mutant of the moss, Physcomitrella patens. After fixation and embedding at low temperature, immunoreactivity was visualized in protonemal filaments of the moss mutant by the use of indirect immunogold labelling. In the mutant, the labelling was predominantly in the wall of the protonemal cells. Neither the wild-type nor control treatments showed any labelling. The signficance of these observations is discussed with respect to the applicability of immunocytochemical techniques for the localization of low-molecular-weight compounds in plant tissue.

title: Isopentenyladenine from mutants of the moss, Physcomitrella patens
abstract: The culture media from gametophore over-producing mutants of the moss Physcomitrella patens have been examined for their cytokinin content. Two cytokinins have been detected, one of which has been identified as N6-(Δ2-isopentenyl) adenine (2iP).

title: Cytokinins from the Moss Physcomitrella patens.
abstract: Gametophore-over-producing mutants of the moss, Physcomitrella patens, when grown in liquid culture export high levels of cytokinin into their culture medium. The cytokinin produced by these mutants is postulated to account for their peculiar phenotype, that of mosses treated with exogenous cytokinin. N(6)-(Delta(2)-isopentenyl)adenine, the major cytokinin, has been identified previously in two of these mutants (Wang, Cove, Beutelmann, Hartmann 1980 Phytochemistry 19: 1103-1105) and now in additional representatives. A second cytokinin, zeatin, has been identified by its chromatographic behavior and mass spectrum including chemical ionization mass spectrometry of its permethyl derivative.

title: Cytokinin Biosynthesis in Mutants of the Moss Physcomitrella patens.
abstract: Three cytokinin-over-producing mutants of the moss, Physcomitrella patens, have been shown to convert [8-(14)C]adenine to N(6)-[(14)C](Delta(2)-isopentenyl)adenine, the presence of which was confirmed by thin layer chromatography, high performance liquid chromatography, and recrystallization to constant specific radioactivity. The labeled cytokinin was detected in the culture medium within 6 hours and the tissue itself appears to contain both labeled N(6)-(Delta(2)-isopentenyl)adenine and N(6)-(Delta(2)-isopentenyl)adenosine monophosphate.

title: The Hormonal Regulation of Protonema Development in Mosses. IV. The Role of Ca2+ as Cytokinin Effector
abstract: Experiments with the moss Funaria hygrometrica were performed to clarify the involvement of Ca during cytokinin dependent bud formation. Protonemata were cultivated either on a Ca 2+ -free or a cobalt or lanthanum medium. Buds were induced with 10 J,M kinetin. A detailed analysis has shown that the reduction of the number of buds is an effect of the diminished number of sites able to bear a bud rather than the inhibition of the bud forming process. The buds produced were unable to undergo cell division and remained round and unicellular. Treatment with CTC showed that the bud-like structures lacked the characteristic apical Ca 2+ -CTC fluorescence. Staining with acridine orange resulted in an identical pattern of fluorescences in both normal and Ca 2+ -free buds. It is concluded that the cytokinin-mediated process of bud formation is not regulated by the external Ca 2+ content. This is in contrast to the Ca 2+ dependent cell division process, which requires much lower concentrations of cytokinin.

title: Tip-localized calcium entry fluctuates during pollen tube growth.
abstract: Studies have been conducted on the dynamics of Ca2+ entry in pollen tubes using ratiometric ion imaging to measure the intracellular gradient and an ion selective vibrating electrode to detect the extracellular influx. A steep tip-focused gradient occurs in all species examined, including Lilium longiflorum, Nicotiana sylvestris, and Tradescantia virginiana. Anlaysis of Lilium pollen tubes loaded with dextran conjugated fura-2 reveals that the gradient derives from Ca2+ entry that is restricted to a small area of plasma membrane at the extreme apex of the tube dome. Since the apical membrane is continually swept to the flanks during tube elongation, either Ca2+ channels are specifically retained at the extreme apex or, as seems more likely, the Ca2+ channels which were active at the tip rapidly inactivate, as new ones are inserted during vesicle fusion. Ratiometric imaging further indicates that the high point of the gradient fluctuates in magnitude from 0.75 to above 3 microM, during measuring intervals of 60 sec, with the elevated points being correlated with an increased rate of tube growth. Independent analysis of the growth at 2- to 3-sec intervals reveals that the rates can fluctuate more than threefold; tubes longer than 700 mu m exhibit oscillations with a period of 23 sec, while tubes shorter than 700 mu m display erratic fluctuations. Inhibition of pollen tube growth caused by mild temperature shock or caffeine (1.5 to 3.0 mM) is correlated with the dissipation of the tip-focused gradient and the Ca2+ influx. Recovery from both treatments is denoted by a global swelling of the pollen tube tip, concomitant with a high transient entry of Ca2+ in the tip. The location of the highest Ca2+ domain within the tip region defines the point from which normal cylindrical elongation will proceed.

title: Growth of Pollen Tubes of Papaver rhoeas Is Regulated by a Slow-Moving Calcium Wave Propagated by Inositol 1,4,5-Trisphosphate.
abstract: A signaling role for cytosolic free Ca2+ ([Ca2+]i) in regulating Papaver rhoeas pollen tube growth during the self-incompatibility response has been demonstrated previously. In this article, we investigate the involvement of the phosphoinositide signal transduction pathway in Ca2+-mediated pollen tube inhibition. We demonstrate that P. rhoeas pollen tubes have a Ca2+-dependent polyphosphoinositide-specific phospholipase C activity that is inhibited by neomycin.

title: Localized Apical Increases of Cytosolic Free Calcium Control Pollen Tube Orientation.
abstract: To reach the ovule, pollen tubes must undergo many changes in growth direction. We have shown in previous work that elevation of cytosolic free calcium ([Ca2+]c) can manipulate orientation in growing pollen tubes, but our results suggested that [Ca2+]c changes either in the tip or in more distal regions might regulate the critical orienting mechanism. To identify the spatial location of the orienting motor, we combined the techniques of ion imaging with confocal microscopy and localized photoactivation of loaded caged Ca2+ (nitr-5) and diazo-2 (a caged Ca2+ chelator) to manipulate [Ca2+]c in different pollen tube domains. We found that increasing [Ca2+]c on one side of the pollen tube apex induced reorientation of the growth axis toward that side. Similarly, a decrease in [Ca2+]c promoted bending toward the opposite side. These effects could be mimicked by imposing localized external gradients of an ionophore (A23187) or a Ca2+ channel blocker (GdCl3); the pollen tubes bend toward the highest concentration of A23187 and away from GdCl3. Manipulation of [Ca2+]c in regions farther back from the apical zone also induced changes in growth direction, but the new orientation was at random. We observed communication of these distal events to the tip through a slow-moving [Ca2+]c wave. These data show that localized changes of [Ca2+]c in the tip, which could result from asymmetric channel activity, control the direction of pollen tube growth.

title: Cytokinin stimulates dihydropyridine-sensitive calcium uptake in moss protoplasts.
abstract: Ca2+ influx through dihydropyridine (DHP)-sensitive Ca2+ channels is thought to be an early event in cytokinin-induced bud formation in moss protonema because DHP antagonists inhibit bud formation in the presence of cytokinin and DHP agonists stimulate bud formation in the absence of cytokinin [Conrad, P. A. & Helper, P. K. (1988) Plant Physiol. 86, 684-687]. In the present study, we established the presence of a DHP-sensitive Ca2+ transport system by measuring 45Ca2+ influx into moss protoplasts. Ca2+ influx was stimulated by external KCl (up to 5 mM), indicating that transport is voltage-dependent. K(+)-induced Ca2+ influx was DHP-sensitive with > 50% inhibition at 500 nM nifedipine. Ca2+ influx was stimulated by increasing concentrations of the DHP Ca2+ channel agonist Bay K8644 with half-maximal effects at 25 nM; this stimulation was seen only in the absence of K+, suggesting that the agonist works preferentially on polarized membranes. Ca2+ influx was also inhibited by phenylalkylamines (verapamil) and benzothiazepines (diltiazem). The phytohormone 6-benzylaminopurine consistently stimulated Ca2+ influx with a Km value of 1 nM, whereas adenine, indoleacetic acid, and gibberellic acid had no effect on Ca2+ transport. The cytokinins kinetin and trans-zeatin caused a greater stimulation of Ca2+ influx and induced more bud formation than did 6-benzylaminopurine. These results indicate that Ca2+ is taken up into moss protoplasts through voltage-dependent DHP-sensitive Ca2+ channels on the plasma membrane and that one of the cytokinin effects in the induction of bud formation is regulation of this plasma membrane Ca2+ channel.

title: 1,4-Dihydropyridine Binding Sites in Moss Plasma Membranes
abstract: An increase in cytoplasmic calcium is an early event in hormone (cytokinin)-induced vegetative bud formation in the moss Physcomitrella patens. Whole cell and calcium transport studies have implicated 1,4-dihydropyridine-sensitive calcium channels in this increase in cellular calcium. To understand the molecular nature of the dihydropyridine-sensitive calcium channel, we have established conditions for the binding of the arylazide 1,4-dihydropyridine, [3H]azidopine, to its receptor in moss plasma membranes. [3H]Azidopine bound specifically in a saturable and reversible manner. The K for [3H]azidopine binding was 5.2 nM and the Bmax was 35.6 pmol/mg of protein. Association and dissociation of the receptor and [3H]azidopine were temperature-dependent, and association varied as a function of pH. Binding was inhibited by dihydropyridine, phenylalkylamine, and benzothiazepine calcium channel blockers, bepridil, lanthanum, and N-ethylmaleimide. [3H]Azidopine binding was stimulated by cations including calcium, strontium, manganese, and barium. [3H]Azidopine binding was also stimulated by cytokinin with a Kvalue for kinetin of 0.13 nM. These studies utilize a simple plant system to provide a biochemical framework for understanding calcium regulation during development and have implications for understanding mechanisms of signal transduction in plants.

title: G Proteins Regulate Dihydropyridine Binding to Moss Plasma Membranes*
abstract: The role of calcium as an activator and regulator of many biological processes is linked to the ability of the cell to rapidly change its cytoplasmic calcium levels. Calcium acts as an intracellular messenger in hormone-induced bud formation during the development of the moss Physcomitrella patens. Calcium transport and ligand binding studies have implicated plasma membrane-localized 1,4-dihydropyridine (DHP)-sensitive calcium channels in this increase in cellular calcium. To understand the regulation of the moss calcium channel, we investigated the involvement of GTP binding regulatory proteins (G proteins). Guanosine 5′-(γ-thio)triphosphate (GTPγS), a nonhydrolyzable GTP analog that locks G proteins into their active state, stimulated DHP binding to high affinity receptors in the moss plasma membrane. DHP binding was measured as the ability of the DHP agonist Bay K8644 or the DHP antagonist nifedipine to compete with the DHP arylazide [3H]azidopine for binding to moss plasma membranes. G protein stimulation of binding was seen when competition was carried out with either nifedipine or Bay K8644. G proteins regulated the rates of association and dissociation of bound [3H]azidopine, and stimulation was dependent on GTPγS concentration. Guanosine 5′-(β-thio)diphosphate, a GDP analog that locks G proteins into their inactivated state, did not affect the dose dependence of either the agonist or the antagonist. These results suggest that G proteins may act via a membrane-delimited pathway to regulate calcium channels in the moss plasma membrane.

title: Structure and function of voltage-gated ion channels
abstract: The principal subunits of the voltage-gated Na+, Ca2+ and K+ channels are members of a related gene family and are functionally autonomous in voltage-dependent activation, ion conductance and inactivation. In this article, recent work locating the structural elements that are responsible for these three basic functions of the voltage-gated ion channels is reviewed. These studies reveal strong functional analogies among the different ion channels and suggest that the striking differences in their properties arise as variations on a common structural and functional theme.

title: Mechanisms of asymmetric cell division: Two Bs or not two Bs, that is the question
abstract: How sister cells can have different fates is a fundamental aspect of the problem of how cell diversity is generated during development. In principle, sister cells that aredifferent could be produced in two distinct ways. First, polar mother cells could divide to generate daughters that are different from the time they are formed. Alternatively, two identical sister cells could be generated and become different as a consequence of some later event. Consideration of these mechanisms raises immediate further questions: What causes a mother cell to be polar? How do initially identical sister cells become different? And in each case, how do initial differences in sister cells lead to their ultimately distinct fates? Answers to these questions would provide important insights into the mechanisms responsible for the control of development. In this review, we use the term “asymmetric cell division” to refer to any cell division in which sister cells have different fates. These distinct fates can be recognized by differences in size, shape, or in other morphological or biochemical features, or, if the sister cells are blast cells, by differences in their subsequent patterns of cell divisions and in the number and nature of the descendant cells they produce. Thus, if a cell division generates sister cells with distinct fates “A” and “B,” that division is considered to be asymmetric . By contrast, if a division generates sister cells with the same fate, e.g., 8, that division is symmetric. In short, the problem of asymmetric cell division is how a cell can divide to produce cells with distinct fates A and B instead of two cells that express the same fate B. As we use the term, an asymmetric cell division need not produce sister cells of different sizes and can produce initially identical cells with fates tllat differ as a consequence of subsequent cell interactions. Our current understanding of the mechanisms responsible for asymmetric cell division derives from a wide variety of experimental approaches using many different organisms. For example, classical observations of animal embryology established that specific regions of egg cytoplasm are partitioned to specific descendant cells that have distinct fates and also that certain substances are localized within the cytoplasm of the egg and can be differentially distributed to daughter cells. More recent studies involving micromanipulation or using a microelectrode to kill specific cells have revealed that cell interactions can cause sister cells to express different fates. Genes that Review

title: The synthesis and biological properties of 8-azido-N6-benzyladenine, a potential photoaffinity reagent for cytokinin
abstract: A cytokinin photoaffinity reagent, 8-azido-N6-benzyladenine (8N3BA), was synthesized from 8-bromoadenosine via azide replacement, benzylation at N−1, rearrangement to the N-6-benzyl derivative and acid hydrolysis. The compound thus obtained was found to have full cytokinin activity in the moss and tobacco cell-suspension bioassays. Photolysis of 8N3BA was accomplished with long and short-wavelength ultraviolet light and produced compounds which had very little or no biological activity in the two bioassays. In-vivo photolysis of 8N3BA caused loss of the cytokinin activity of this compound in moss protonemata. This result was similar to earlier ones where the biological response of moss protonemata to benzyladenine was reversed following removal of the hormone by a short rinse with water.

title: Complementation analysis of auxotrophic mutants of the moss, Physcomitrella patens, using protoplast fusion
abstract: SummaryFusion of protoplasts from the moss, Physcomitrella patens, was induced using polyethyleneglycol. Protoplasts were isolated from six nicotinic acid auxotrophic strains of independent origin and fusion was induced in all possible pairwise combinations. Complementation was detected by the ability to recover hybrids able to grow without nicotinic acid supplement. On the basis of the results presented, three nonoverlapping complementation groups were identified.

title: Stable transformation of the moss Physcomitrella patens
abstract: SummaryWe report the stable transformation of Physcomitrella patens to either G418 or hygromycin B resistance following polyethylene glycol-mediated direct DNA uptake by protoplasts. The method described in this paper was used successfully in independent experiments carried out in our two laboratories. Transformation was assessed by the following criteria: selection of antibiotic-resistant plants, mitotic and meiotic stability of phenotypes after removal of selective pressure and stable transmission of the character to the offspring; Southern hybridisation analysis of genomic DNA to show integration of the plasmid DNA; segregation of the resistance gene following crosses with antibiotic-sensitive strains; and finally Southern hybridisation analysis of both resistant and sensitive progeny. In addition to stable transformants, a heterogeneous class of unstable transformants was obtained.

title: Calcium-sensitive cls4 mutant of Saccharomyces cerevisiae with a defect in bud formation
abstract: A calcium-sensitive cls4 mutant of Saccharomyces cerevisiae ceased dividing in the presence of 100 mM CaCl2, producing large, round, unbudded cells. Since its DNA replication and nuclear division still continued after interruption of normal budding, the cls4 mutant had a defect in bud formation in Ca2+-rich medium. Its calcium content and calcium uptake activity were the same as those of the wild-type strain, suggesting that the primary defect of the mutation was not in a Ca2+ transport system. Genetic analysis showed that the cls4 mutation did not complement the cdc24-1 mutation, which is known to be a temperature-sensitive mutation affecting bud formation and localized cell surface growth at a restrictive temperature. Moreover, cls4 was tightly linked to cdc24, and a yeast 3.4-kilobase-pair DNA fragment carrying both the CLS4 and CDC24 genes was obtained. These results suggest that the cls4 mutation is allelic to the cdc24 mutation. Thus, Ca2+ ion seems to control bud formation and bud-localized cell surface growth.

title: T‐DNA insertion mutagenesis in Arabidopsis: mutational spectrum
abstract: More than 8000 transformants of Arabidopsis have been generated by treating germinating seeds with cultures of Agrobacterium tumefaciens. Genetic characterization of a subset of the transformants indicates that they contain an average of 1.4 inserts each, as assayed by kanamycin resistance. Molecular analysis shows that the inserts are predominantly concatamers of T-DNAs arranged as direct and inverted repeats. More recently these 8000 lines have been screened under a variety of growth conditions for visible alterations in phenotype. More than 1000 putative mutants were observed during the application of these screening procedures. These mutants fall into several general classes: seedling-lethals, size variants, pigment, embryo-defective, reduced-fertility, dramatic (morphological), and physiological. The majority of the mutants (88%) segregate in a Mendelian manner for the mutant phenotype. An analysis of approximately 50 mutants in this group shows that > 80% are tagged with a functional insert. The wide spectrum of mutants observed suggests that it may be feasible to develop a comprehensive collection of mutant lines in which each gene is tagged by a T-DNA insertion.