Germination of Monocolpate Angiosperm Pollen: Effects of Inhibitory Factors and the Ca2+-Channel Blocker, Nifedipine

Hydration of pollen of Narcissus pseudonarcissus was retardedand germination blocked in media with supra-optimal concentrationsof osmoticum. Activation of the grains, expressed in circulatorymovement in the vegetative cell, was not blocked. Wall developmentwas disrupted, and pectic material and callose were depositedthroughout. In the absence of calcium many grains burst on hydration.The survivors showed evidence of activation, but few tubes wereformed. In medium with supra-optimal Ca²⁺, activation proceeded,but where tube tips were produced they became occluded withcallose, which eventually formed a general lining to the intine.Nifedipine, a Ca²⁺-blocker, did not prevent activation at 10^–4M, but reduced callose deposition and inhibited polarized movementin the vegetative cell. Prominences formed at the germinationsites were mostly low and rounded. During recovery in normalmedium, tube tips with normal callose linings were formed. Colchicine,a microtubule inhibitor, had no effect on activation or germination.Cytochalasin D, an actin inhibitor, prevented activation ofthe vegetative cell, but did not arrest all wall deposition.Movement began soon after transfer to normal medium, and somegrains produced adventitious tube tips. While Ca²⁺ appears notto be essential for activation, these results may be interpretedas indicating links in the normal course of germination betweenthe initial Ca²⁺ influx at the potential germination sites and:(a) polarization of movement in the vegetative cell, probablyrelated to re-orientation of the actin cytoskeleton; and (b)patterned deposition of callose, which appears to have an importantmorphogenetic role. Narcissus pseudonarcissus, pollen activation, pollen germination, osmotic effects, actin cytoskeleton, nifedipine, cytochalasin D, colchicine, role of Ca²⁺ flux     

The Comportment of the Vegetative Nucleus and Generative Cell in the Pollen and Pollen Tubes of Helleborus foetidus L

It has been reported that in species of Plumbaginaceae, Chenopodiaceae,Cruciferae and Amaryllidaceae a ‘male germ unit’is formed in which the two male gametes remain inter-connected,with one of the pair linked intimately to the vegetative nucleus.In two species the unit has been shown to remain intact in thepollen tube, and some accounts imply that it is polarized inits movement, the vegetative nucleus leading in the tube. Evidence given in this paper indicates that such a unit is unlikelyto be present in Helleborus foetidus L. (Ranunculaceae). Applicationof an optical sectioning technique has shown that at no timeis there a persistent linkage between the generative cell andthe vegetative nucleus in unhydrated, hydrated and germinatingpollen, nor is one present in the early pollen tube. Furthermore,no inter-connections between the two entities were seen in protoplastsfrom living, hydrated and incipiently germinating grains isolatedmechanically in an osmotically balancing medium. Following germination,the vegetative nucleus leaves the grain in advance of the generativecell in most instances, but in the samples examined the generativecell led in about 30 per cent of the tubes. Assembling a polarisedmale germ unit in these circumstances would require (a) theformation of an inter-connection between the vegetative nucleusand the generative cell or one of the gametes derived from itduring passage through the tube, and (b) where the generativecell initially leads in the tube, an exchange in relative positions.It is considered improbable that these conditions could consistentlybe met. Mature, incipiently germinating pollen of H. foetidus releasesa fibrillar component when extruded into suitable media. Websor clusters of fibrils are commonly seen to be associated withboth the vegetative nucleus and the generative cell. The possibilitythat the fibrils are composed of aggregates of microfilamentsis considered. Helleborus foetidus L., pollen germination, generative cell, vegetative nucleus, male germ unit     

Sites of Origin of the Peripheral Microtubule System of the Vegetative Cell of the Angiosperm Pollen Tube

From earlier published work it is known that microtubules inthe vegetative cell of angiosperm pollen tubes mainly occurin the form of longitudinally disposed strands closely associatedwith the plasmalemma. This peripheral cytoskeleton is extendedapically at a speed matching the growth rate of the tube. Immunofluorescencelocalization shows that in the actively elongating tube it originatesin the sub-apical zone in bands or ribbons up to 2 µmwide, interpreted here as comprising aggregates of apposed,axially oriented microtubules. These appear first in the corticalcytoplasm in close association with the wall in the part ofthe tube where the callose inner lining can first be detected.The bands do not extend apically into the region of the pecticsheath of the extreme tip. In the course of normal growth, theperipheral microtubule investment remains in the older partsof the tube from which the bulk of the cytoplasm has been withdrawn,indicating that tubulin is probably not recycled. If the growth of the tube is retarded, the inner callosic layerextends apically. The acropetal movement of callose is accompaniedby a migration of the limit of detectable tubulin towards theextreme tip, and the axially oriented bands are replaced bya confused mass of granules and short spicules. It is suggested that the bands represent nucleation zones associatedwith the stabilizing plasmalemma in the sub-apical stretch ofthe tube where the insertion of wall-precursor material is diminishing,and that it is from these zones that the microtubule cytoskeletonof the pollen tube originates. Since during growth the nucleationzones progress rapidly forward into association with new membrane,it is considered unlikely that their sites are determined bylocal differentiations of the plasmalemma. An alternative possibilityis that the distribution of the zones is related to the calciumion gradient known to be present in the apical stretch of theextending pollen tube. Microtubules, pollen tube growth, Lilium auratum     

Tubulin and Male-gamete Interconnections in the Pollen Tubes of the Grass Alopecurus pratensis

Tubulin was localized in pollen tubes of the grass Alopecuruspratensis by immunofluorescence using a rat monoclonal yeastantitubulin as a primary antibody. In tubes fixed in activegrowth with paraformaldehyde fixative and permeabilized withdetergent, the two male gametes were found to be linked by tubulin-containingcytoplasmic bridges. The connections appeared to persist throughoutthe growth of the tube, but they were highly mutable, the distancebetween the gamete pair varying greatly, the gametes sometimesactually overlapping in the tube. Systems of longitudinally disposed, discrete microtubule strandscomparable with those seen in generative cells of species withbicellular pollen grains were not observed in the gametes. However,an envelope of antitubulin-binding material was apparent atthe cell boundary in many instances, possibly to be interpretedas a sheath of closely packed microtubules seen in profile.Irregular masses, probably representing pools of unpolymerizedor partly polymerized tubulin, were also present in most ofthe gametes. Alopecurus pratensis, tubulin, pollen tubes, grass male gametes     

A Cytochemical Study of DNA, RNA, and Protein in the Developing Maize Anther: II. Observations

Changes in acetic-alcohol fixable DNA, RNA, and protein werefollowed in the tapetum, sporogenous tissue, and spores of thedeveloping maize anther using standard cytochemical methodsand microdensitometry. In the tapetum, early nuclear divisionsoccur without prior DNA synthesis, giving a population of IC nuclei. Subsequent synthesis produces the equivalent of 34,000C amounts per pollen sac, 20 times more than is present in thespores before pollen mitosis. The main tapetal RNA synthesisis during the meiotic prophase, with a further period of accumulationin the interval, tetrad to young spores. In the meiocytes, theprincipal accumulation is in the early prophase, with no synthesisduring the meiotic divisions or through the tetrad period. Proteinaccumulation occurs in the tapetum up to mid-meiotic prophase;after this there is a pause, followed by further synthesis frommeiotic metaphase I to the final dissolution of the tissue.In the meiocytes, protein is accumulated through the early prophase;there is no synthesis during the meiotic mitoses or in the tetradperiod, but active accumula-tion occurs in the developing spores. The implications of these observations are discussed in relationto the function of the tapetum.     

Effect of 2-Thiouracil on Cell Differentiation and Leaf Morphogenesis in Cannabis sativa

When supplied through the leaf at low dosage levels, the pyrimidineanalogue, 2-thiouracil, progressively arrests apical growthand organogenesis in young plants of Cannabis sativa. Duringthe treatment period, a generation of leaves arises in whichcell differentiation is inhibited without a parallel reductionin laminal expansion. With the withdrawal of treatment, apicalgrowth is resumed, but severe morphogenetic defects later becomeevident in leaves developing from primordia initiated duringthe treatment period or shortly afterwards, and there is simultaneouslya loss of apical dominance and of the control of phyllotaxis.Growth of a more normal pattern is, however, gradually restoredat stem apices, although local anomalies in the anatomy of thelamina recur throughout the life of the plant, and otherwisenormal leaves continue to show sporadic flecks or streakingsof abnormal pigmentation. The latter phenomenon suggests thatthe analogue induces a persistent state of instability in someof the proplastids of meristematic tissues.     

Microsporogenesis in some Triploid Dactylorchid Hybrids

During meiosis in naturally occurring triploid hybrids betweenthe diploid Orchis fuchsii Druce (2n = 4O) and the two tetraploids,O. purpurella Steph. and O. praetermissa Druce (2n = 8O), thereis a regular formation of 20 bivalents and 20 bivalents. Sincethe two tetraploid species themselves show typical ‘diploid’behaviour in synapsis and fertility, they are considered tobe allopolyploids, and the hybrid pairing to be allosyndetic.The implication is therefore that both tetraploids are amphidiploidsof which O. fuchsii has been one progenitor. It is suggestedthat varieties of the polytypic diploid O. latifolia L. sec.Pugsl. may have been the other progenitors. A feature of interestin the microsporogenesis of both parents and hybrids is theclose synchronization of nuclear events in the pollen massulae,which behave as physiological units throughout meiosis and pollen-mitosis.In the triploids, although numerous dysploid nuclei are produced,none dies prematurely, probably because of mutual compensationwithin what is, in effect, a common cytoplasmic matrix.     

The Pollen-stigma Interaction: Pollen-tube Penetration in Crocus

In a compatible pollination in Crocus, pollen tube tips enterthe stigma papillae after the enzymic erosion of the cuticle,and the tubes continue downward growth towards the ovary betweenthe cuticle and the underlying pectocellulosic wall. The cuticleof the receptive zone of the stigma papilla is chambered, thechambers containing a secretion accumulated during the maturationof the stigma. Pollen exudates contain various acid hydrolases,but are incapable alone of eroding stigma cutin. Furthermore,there is no penetration when the proteins of the wall-held stigmasecretions are degraded enzymically. These facts are taken toindicate that the pollen contributes a ‘cutinase’precursor which is activated by a factor or factors held inthe stigma secretion. Pollens of certain Cruciferae producetubes capable of penetrating the Crocus stigma cuticle, suggestingthat notwithstanding the taxonomic remoteness of Cruciferaeand Iridaceae the enzyme activation systems are quite similar.