QUANTITATIVE STUDY OF DEVELOPMENT OF THE GIANT COENOCYTE,CAULERPA PROLIFERA

The lengths of rhizomes, rhizoid clusters, and leaves of many plants of Caulerpa prolifera cultured under controlled conditions were measured daily. Their rates and patterns of growth were analyzed mathematically with the aid of an electronic computer. All organs elongated at the rate of ca. 4.4 mm per day. Rhizomes elongated linearly for more than 2 weeks, and curvilinear regression usually did not improve the fit significantly. Rhizome elongation is apical. Every 1.18 days, on the average, a rhizoid cluster developed on the lower surface of each rhizome. The length of the younger rhizoid clusters shows a linear relationship to their distance from the rhizome tip. Little or no significant improvement of fit resulted from fitting polynomials of the 2nd through 4th degree. Accordingly, by extrapolating the straight line to zero rhizoid length, we calculated the presumptive site of rhizoid initiation to be 0.84 mm from the rhizome tip. The rate and distribution of growth were compared to those of other coenocytes and of multicellular plants.     

Effects of continuous illumination on the rhizoid cluster of Caulerpa prolifera

Under a photoperiodic regime of 12-hr light and 12-hr dark (12L-12D)and continuous illumination (24L-0D), at 24.0±1°C,the rate of elongation of the rhizoid cluster was 8.1 mm/day,and the presumptive site of the cluster initiation was 1.1–1.9mm from the rhizome tip. However, the plastochron interval under24L-0D regime was shorter (0.87 day) than under 12L-l2D regime(1.18 days). Under both regimes, the plants tended to adjusttheir presumptive time of cluster initiation to be in the twoperiods of the day, 0400–0900 hr and 1800–2100 hr.None of the following seemed to correlate with the initiationof a new cluster: a certain number of hours after the initiationof the youngest cluster, a certain distance on the rhizome distalto the youngest cluster, the youngest cluster reaching to acertain length, or a certain value of the rhizoid plastochronindex. This adjustment by the plants thus suggested the plantshaving an ability to perceive the two preferred periods forcluster initiation. ¹ Report of work supported by research grant GB-7885 from theNational Science Foundation, and in part by Research Councilof Rutgers University. (Received June 2, 1972; )     

Effects of continuous illumination on the rhizome of Caulerpa prolifera

Caulerpa plants were grown under a 12-hr light and 12-hr dark(12L-12D) regime for 8 days followed by 8 days of continuouslight (24L-0D). Under both light regimes the elongation of therhizome was by means of "tip growth". However, the rate of rhizomeelongation in 24L-0D regime (10.9 mm/day) was higher than thatof 12L-12D regime (7.9 mm/day). Jn the region of 1.5 mm fromthe tip, the RERE (relative elemental rate of elongation) under24L-0D and 12L-12D regimes were respectively 4% and 3% elongationper hr. Under 12L-12D photoperiod the subapical part of therhizome exhibited distinctive oscillation: bending upward duringthe light period or at the time of rhizoid cluster initiation,and in the dark becoming relatively straight except at the timeof cluster initiation. No such distinctive oscillation was observedin continuous light. ¹ Report of work supported by research grant GB-7885 from theNational Science Foundation, and in part by Research Councilof Rutgers University. (Received June 8, 1971; )     

Rhizome gravitropism precedes gravimorphogenesis after inversion of the green algal coenocyte Caulerpa prolifera (Caulerpales)

Previous investigations reported that the marine alga Caulerpa prolifera, a giant coenocyte, had a fast morphogenetic response to a change in position with regard to gravity: the next rhizoid formed approximately 1 day later on the new underside of the inverted rhizome (Jacobs and Olson, 1980, American Journal of Botany 67: 141–146). Preceding the change in site of rhizoid development was a striking accumulation of amyloplasts at the new prospective rhizoid-initiation site in the rhizome tip. Detailed monitoring with video equipment of inverted Caulerpa plants, growing under controlled conditions, now reveals that after the amyloplast accumulation, but before the gravimorphogenetic effect on rhizoid development, the inverted rhizome tip shows a negative gravitropism that restores it to its normally upturned position. Because the experiments were performed with two-sided illumination, this was clearly not a phototropic response.     

DEVELOPMENTAL CHANGES IN THE ALGAL COENOCYTE CAULERPA PROLIFERA (SIPHONALES) AFTER INVERSION WITH RESPECT TO GRAVITY

The algal coenocyte Caulerpa prolifera produces three types of organs, each with a different orientation. The effect of gravity in controlling the development of these organs was investigated. We inverted plants by rotating them 180° around the horizontal rhizome axis, then compared development of the inverted plants with plants matched by size and differentiation. Quantitative data were obtained from photographic records. After inversion the site of organ differentiation was changed with no change in the timing: the next rhizoid (and all subsequent ones) differentiated on the “now-under side” of the inverted rhizome, the next leaf formed on the “now-upper side.” Despite the fast change in the site of organ differentiation, other parameters were not changed by inversion (e.g., rate of elongation of rhizomes or leaves, rate of formation of leaves or rhizoids). Because such changes also occurred in plants with balanced lighting from two sides, it is clear that gravity alone can control these changes without reinforcement from top illumination. After leaves were initiated, they did not change their orientation, showing neither positive phototropism (at our light levels) nor negative geotropism, even when elongating substantially. Torsion of the rhizome tip did not precede the change in site of rhizoid initiation.     

CALCIUM DELAYS THE ONSET OF CELL DIVISION AND RHIZOID ELONGATION IN GEMMAE OF THE CALCIFUGE FERN,VITTARIA GRAMINIFOLIA

Vittaria graminifolia gametophytes reproduce asexually by linear six-celled propagules known as gemmae. It has been shown previously that asymmetric cell division and rhizoid elongation in gemmae are inhibited by millimolar concentrations of Ca^{+ +}. The present paper shows that millimolar Ca^{+ +} delays the onset of cell division at a point prior to mitosis, without affecting the maximal rate of cell division. Ca^{+ +} is most effective in delaying cell division when it is present during the first 24 hours of culture, 2 or 3 days before the initiation of cell division. Millimolar Ca^{+ +} inhibits rhizoid elongation by delaying its onset, while also reducing its maximal rate. Ca^{+ +} is also most effective in delaying rhizoid elongation during the first 24 hours of culture. Culture of gemmae on Ethylglycol-bis-(aminoethyl ether)-N,N,N‘,N‘-tetraacetic acid-buffered media shows that the maximum frequency of cell division occurs at pCa 7, while for the initiation of rhizoid elongation, it occurs at pCa 5.     

Accumulation of amyloplasts on the bottom of normal and inverted rhizome tips of Caulerpa prolifera (Forsskål) Lamouroux

Caulerpa prolifera (Chlorophyta) exhibits a gravimorphogenetic response to inversion by switching the site of new rhizoid initiations to correspond with the new direction of the gravity stimulus. When plants were fixed at 6 and 24 h after being held in either a normal or an inverted position the switch in the site of organ differentiation, upon inversion, was found to be preceded by the accumulation of starch-containing amyloplasts on the bottom of the rhizome. Approximately 1.5–2.0 times as many amyloplasts were found at the bottom of normal or inverted rhizomes as compared with the top in a region extending from 200 to 1,000 μm behind the rhizome tip. All new rhizoid initials were located in the region of amyloplast accumulation and each rhizoid initial contained numerous amyloplasts.     

DEVELOPMENTAL STUDIES ON THE COENOCYTIC ALGA, CAULERPA SERTULARIOIDES

Thallus growth and development in the coenocyti alga Caulerpa sertularioides (Gmelin) Howe have been studied quantitatively. In unsupplemented seawater at 26 C and in a 12:12 hr light/dark cycle, new rhizomes formed near the old growing points on thalli transplanted from the ocean. Adjacent to the apices of new rhizomes, rhizoids were produced downward, regularly spaced and at a rate of about 1.5/day. Upright. shoots developed irregularly in acropetal succession behind the tips of either the parent or the new rhizomes, which arose as branches of the old. Rates of rhizome, rhizoid, and upright shoot elongation were 0.4, 0.81, and 0.54 cm/day, respectively. Thalli survived up to 2 months in unsupplemented seawater. Long-term growth was obtained by varying culture conditions. A substratum of sand, apparently rich in microorganisms, produced long-term thallus growth in seawater, and the form of development changed so that upright shoot formation was promoted and rhizome elongation halved. Similar effects were elicited by indole-3-acetic acid, 5 × 10^-5, M in seawater and by sap expressed from C. racemosa or C. sertularioides and added to seawater at 2.5–10% by volume. The regulation of development in an algal coenocyte is discussed and analogies with regulation in multicellular plants are drawn.     

LIGHT-DEPENDENT INHIBITION OF CELL DIVISION AND RHIZOID ELONGATION IN GEMMAE OF THE FERN,VITTARIA GRAMINIFOLIA

Gametophytes of the shoe-string fern Vittaria graminifolia produce linear, six-celled propagules called gemmae. The terminal cells of each gemma elongate into primary rhizoids in culture, and the inner body cells divide asymmetrically to produce prothallial or rhizoid initials. The initiation of both asymmetric cell division and rhizoid elongation is delayed by light intensities greater than 2 w/m². The maximal rates of cell division and rhizoid elongation are unaltered. A 24-hr pulse of high light intensity delays cell division and rhizoid elongation to the same extent, whenever applied during the first 3 d of culture. The model we propose for cell division hypothesizes the existence of a preparatory phase of finite duration prior to mitosis that is sensitive to light intensity. If a cell is irradiated by light intensities greater than 2 w/m² while in the preparatory phase, its entrance into mitosis is delayed. A similar model is proposed for the initiation of rhizoid elongation. Despite the fact that both cell division and rhizoid elongation are dependent on photosynthesis, direct measurements of CO₂-uptake rates show that the inhibitory effects of high light intensities are not due to an inhibition of photosynthesis.     

The ultrastructure of the cell wall of normal and apolar embryos ofFucus vesiculosus

Summary Structure and composition of the walls of normal and apolar embryos ofFucus vesiculosus L. were studied. Fucoidin was found in an amorphous outer layer and in an inner fibrillar layer of the wall, mainly at the rhizoid pole. Also in apolar embryos this inner layer was present; it was markedly thickened at the presumptive site of rhizoid formation.We suggest that initiation and extension of the rhizoid is accompanied by apposition of new fibrillar wall material containing sulphated polysaccharides on the inner side of the wall at the rhizoid pole. In apolar embryos this material accumulates at this pole.     

Localization of membrane-associated calcium during development of fucoid algae using chlorotetracycline

During the first day of development, fertilized eggs of fucoid algae generate an embryonic axis and commence rhizoid growth at one pole. Using Fucus distichus (L.) Powell, F. vesiculosus L. and Pelvetia fastigiata (J.Ag.) DeTony we have investigated the role of calcium in axis formation and fixation as well as in tip growth. The intracellular distribution of membrane-associated calcium was visualized with the fluorescent calcium probe chlorotetracycline (CTC). Punctate fluorescence associated with organelle-like structures was found in conjunction with diffuse staining at all developmental stages. This membrane-associated calcium remained uniformly distributed throughout the cortical cytoplasm while the axis was established, but increased in the rhizoid protuberance at germination. In subsequent development, fluorescence was restricted to the cortical cytoplasm at the elongating tip and at sites of crosswall biosynthesis.The requirement for Ca²⁺ uptake during development was investigated through inhibition studies; influx was impaired with transport antagonists or by removal of extracellular calcium. Both treatments curtailed germination and tip elongation but had little effect on axis polarization. Reductions in external calcium that interfered with elongation also markedly reduced the apical CTC fluorescencence, indicating that calcium uptake and localization are prerequisites for tip growth. This apical Ca²⁺ is probably involved in the secretory process that sustains tip elongation. By contrast, calcium was not implicated in the generation of an embryonic axis.Abbreviations ASW artificial seawater - CTC chlorotetracycline - DU developmental unit - EGTA erhylene glycol bis(amino-ethyl ether) N,N,N¹,N¹–tetraacetic acid - NPN N-phenyl-1-napthylamine     

Cytochalasin treatment disrupts the endogenous currents associated with cell polarization in fucoid zygotes: studies of the role of F-actin in embryogenesis

We determined the distribution of F-actin in fucoid (Pelvetia, Fucus) embryos with nitrobenzoxadiazole-phallacidin, and studied the effect of cytochalasin upon the endogenous currents associated with cell polarization by using the vibrating probe. F-actin is not localized at the presumptive rhizoid immediately after experimental induction of the polar axis with a light gradient; however, a preferential distribution of F-actin develops at the presumptive rhizoid by the time the position of the polar axis is fixed. F-actin continues to be localized at the tip of the rhizoid after germination, except during cytokinesis, when the furrow is the only brightly staining region of the embryo. Incubation with cytochalasin can result in either an enhanced or a diminished pool of F-actin in the embryonic cortex (see Results). Cytochalasin D (100 micrograms/ml) significantly reduces the inward current at the rhizoid pole (n = 11) after a 2.5-h incubation. This drop is concentration dependent and occurs within approximately 30 min at 100 micrograms/ml and approximately 60 min at 10 micrograms/ml. Cytochalasin treatment eliminates the pulsatile component of the current. Preliminary results suggest that 100 micrograms/ml cytochalasin D prevents development of inward current at the presumptive rhizoid but does not completely delocalize this locus if added after photopolarization. We conclude that microfilaments are required for the establishment and maintenance of the pattern of endogenous currents observed during early embryogenesis. This suggests a new model for axis formation and fixation.     

ORGANOGRAPHY,BRANCHING, AND THE PROBLEM OF LEAF VERSUS BUD DIFFERENTIATION IN THE VINING EPIPHYTIC FERN GENUS MICROGRAMMA

Investigation of the development and organography of the shoot systems of Microgramma vacciniifolia and M. squamulosa was undertaken for the purpose of determining: (1) the features of shoot growth that are responsible for the distinctive vining character of these epiphytic ferns; and (2) the mode of origin of branches and their contrast with leaf initiation. Shoots of both species are dorsiventral and plagiotropic (i.e., parallel to the substrate) in habit. Since the shoot apical meristem is radial in transectional symmetry, shoot dorsiventrality in Microgramma is a postgenital or secondary developmental event, and its inception is related to the initiation of lateral appendages. Leaves and buds arise in a distichous phyllotaxis and occupy opposite and alternating positions on the dorsal surfaces and flanks of the rhizome. Endogenous roots are initiated in two rows from the ventral surface of the stem, in the vicinity of the rhizome meristem; however, they do not emerge from the rhizome until some distance behind the tip and do not elongate until the region of substrate contact. We conclude that the vining nature of this fern rhizome is a result of precocious internodal elongation and the concomitant delay of leaf and bud expansion in the region of stem elongation. In addition, observation of branch origin confirms previous suggestions that branching in Microgramma is strictly lateral and extra-axillary and not a dichotomous derivative as proposed by some workers. Leaf and bud primordia differ not only in the nature of their respective vascular supplies but also in their actual course of initiation. In the case of the leaf, the primordium is precociously emergent and exhibits a lenticular apical cell at its summit when it is only one plastochron removed from the flanks of the apical meristem. By contrast, initials of the bud primordium divide less actively and remain in a sunken position for at least 5–6 plastochrons; only when the bud apex becomes expanded and emergent does a tetrahedral apical cell become recognizable at the tip of the bud promeristem. Because of the distinctive pattern of branch and leaf origin, as well as the lack of adventitious and phyllogenous origin of branch primordia, we suggest that the shoot of Microgramma is a useful test organism for the re-examination of the problem of leaf and bud determination in the ferns.     

Band structure of surface electric potential in growing roots

For growing roots of azuki bean (Phaseolus chrysanthos), an electric potential is measured minutely along the surface of the root, together with the surface pH. It was found that the root begins to display a band-type pattern of potential with a spatial period of about 2 cm in a mature region as soon as it grows to about 10 cm in root length, while the surface potential shows only one convex peak around a position 5-20 mm behind a root tip and a succeeding concave peak around 20-35 mm, providing the length of root is shorter than about 10 cm. Since the surface potential takes a relatively positive value on average at the side of the root base compared with that in an elongation zone near the tip, electric current is supposed to flow into the elongation zone, accompanied by some local current loops in the mature region. The present band-type pattern observed first in multi-cell systems seems to be a kind of dissipative structure appearing far from equilibrium, and hence its relationship to growth is discussed.     

Effects of Gibberellic Acid on the Rhizome and Rhizoids of the Algal Coenocyte, Caulerpa prolifera, in Culture

Treatment of the large coenocyte Caulerpa prolifera with a hormonallevel of GA₂ resulted in increased elongation of the rhizomeand more frequent initiation of rhizoids when the hormone wasadded to the Erdschreiber culture medium. No effect of GA₂ wasfound on the rate of initiation or elongation of blades. Theseresults suggest that the recently discovered gravimorphogeneticeffect on rhizoid development is mediated by endogenous gibberellin. Caulerpa prolifera, coenocyte, green alga, gibberellic acid     

Epidermal Cell Division and the Coordination of Leaf and Tiller Development

Initiation and development of grass leaves and tillers are oftendescribed individually with little attention to possible interrelationshipsamong organs. In order to better understand these interrelationships,this research examined epidermal cell division during developmentaltransitions at the apical meristem of tall fescue (Festuca arundinaceaSchreb.). Ten seedlings were harvested each day for a 9-d period,and lengths of main shoot leaves and primary tillers were measured.In addition, numbers and lengths of epidermal cells were determinedfor 0·5 mm segments along the basal 3 mm of each leafand tiller. Primordia development and onset of rapid leaf elongationwere characterized by an increase in the number of cells perepidermal file with mean cell length remaining near 20 µmper cell. After the leaf had lengthened to 1-1·5 mm,cells near the leaf tip ceased dividing and increased in length,at which time leaf elongation rate increased rapidly. Liguleformation, marking the boundary between blade and sheath cells,occurred prior to leaf tip emergence above the whorl of oldersheaths, while the earliest differentiation between blade andsheath cells probably began when leaves were < 1 mm long.Major transitions in leaf and tiller development appeared tobe synchronized among at least three adjacent nodes. At theoldest node, cessation of cell division in the leaf sheath wasaccompanied by initiation of cell division and elongation inthe associated tiller bud. At the next younger node the ligulewas being initiated, while at the youngest node cell divisioncommenced in the leaf primordium, as elongation of a new leafblade began. This synchronization of events suggests a key rolefor the cell division process in regulating leaf and tillerdevelopment.Copyright 1994, 1999 Academic Press Festuca arundinacea Schreb., tall fescue, cell division, leaf initiation, tillering, ligule development     

Separate de Novo and Salvage Purine Pools Are Involved in the Biosynthesis of Theobromine but Not Caffeine in Leaves of Coffea arabica L

We used a video digitizer system to (a) measure changes in the pattern of longitudinal surface extension in primary roots of maize (Zea mays L.) upon application and withdrawal of auxin and (b) compare these patterns during gravitropism in control roots and roots pretreated with auxin. Special attention was paid to the distal elongation zone (DEZ), arbitrarily defined as the region between the meristem and the point within the elongation zone at which the rate of elongation reaches 0.3 of the peak rate. For roots in aqueous solution, the basal limit of the DEZ is about 2.5 mm behind the tip of the root cap. Auxin suppressed elongation throughout the elongation zone, but, after 1 to 3 h, elongation resumed, primarily as a result of induction of rapid elongation in the DEZ. Withdrawal of auxin during the period of strong inhibition resulted in exceptionally rapid elongation attributable to the initiation of rapid elongation in the DEZ plus recovery in the main elongation zone. Gravistimulation of auxin-inhibited roots induced rapid elongation in the DEZ along the top of the root. This resulted in rapid gravitropism even though the elongation rate of the root was zero before gravistimulation. The results indicate that cells of the DEZ differ from cells in the bulk of the elongation zone with respect to auxin sensitivity and that DEZ cells play an important role in gravitropism.     

Apical Structure of Actively Growing Fern Rhizoids Examined by DIC and Confocal Microscopy

The development and cytology of gametophyte primary rhizoidsof the fern Dryopteris affinis was examined using actively growingmaterial. During development an apical cytoplasmic ‘ accumulation’forms and is associated with active tip growth. This accumulationdeteriorates as terminal differentiation and cessation of growthapproaches. During early development the nucleus moves fromthe rhizoid cell base into the newly extending rhizoid. Later,during the active elongation phase, the nucleus takes up a relativelystable location approx. 100 µm behind the extending apex.Towards terminal differentiation the nucleus lags further behindthe tip. In actively growing rhizoids four distinct zones weredistinguished: a richly cytoplasmic ‘cap’; an apicalregion with tubular vacuolar intrusions; a region distinguishedby a peripheral sheath of cytoplasm and fine irregular cytoplasmicstrands connecting to the nucleus; and the main subapical vacuole.Confocal microscopy of gametophytes stained with fluorescentvital dyes, not previously used to examine fern rhizoid structure,confirmed that the tubular vacuolar system extends well intothe apical cytoplasm, and that the network of fine cytoplasmicstrands leads back from the apical cytoplasm to the nucleus.It also revealed that mitochondria are distributed throughoutthe rhizoid and are not excluded from the extreme apex. Membranestaining by FM 4-64 suggested a high density of membrane vesicleswithin the cytoplasm of the extreme apex. Uptake of this endocytosismarker into endomembranes also suggested rapid plasma membraneturnover in the rhizoid. This study highlights the similarityin the developmental stages and appearance of D. affinis rhizoidsto angiosperm root hairs and their much less distinct apicalzonation compared to pollen tubes. Copyright 2000 Annals ofBotany Company Rhizoid, root hair, confocal imaging, vital stains.     

Synthesis of protein and RNA for initiation and growth of the protonema during germination of bracken fern spore

Cycloheximide inhibited initiation and elongation of the protonemal cell during germination of the spores of bracken fern. Incorporation of ¹⁴C-leucine into protein was also profoundly affected by the drug. Concentration of actinomycin D sufficient to inhibit incorporation of ³Huridine into heavy RNA fractions of spores did not prevent initiation of the protonema, but inhibited its subsequent elongation. Protein synthesis during initiation and growth of protonema was not appreciably sensitive to actinomycin D. As in the case of rhizoid initiation, protein synthesis necessary for initiation of protonema during germination appears to involve preformed messenger RNA.