Pollen Wall Ontogeny of Felicia muricata (Asteraceae: Astereae)

Pollen wall development in Felicia muricata Thunb. Nees (Asteraceae)was investigated by electron microscopy after addition of thelypophilic dye malachite green to the primary fixative. Pollenwall formation commences with the deposition of fibrillar electrondense units which represent future interbacular spaces. Radiallyarranged trilammelate structures develop between the fibrillarunits. Sporopollenin is deposited in the trilammelate structures.After release of the microspores from the tetrad, formationof interbacular spaces is accompanied by disintegration of fibrillarunits. Endexine formation occurs on white lines of unit membranedimensions. Intine formation follows the pattern described formost angiosperms. Observation of bead-like globules and pollenkittwith a membraneous structure is presumed to be related to theaddition of malachite green to the fixative.Copyright 1993,1999 Academic Press Felicia muricata (Thunb.) Nees, pollen, pollenkitt, ultrastructure, fixation     

A Persisting Cellulosic Wall of Microspore Mother Cells during Microsporogenesis in Allium tuberosum Rottl. and Cyclamen persicum Mill

Light microscopic studies were carried out on the anthers ofAllium tuberosum Rottl. and Cyclamen persicum Mill. Callosedeposition is initiated within the primary wall round each microsporemother cell during early prophase of meiosis I and is of maximumthickness at the tetrad stage. The original cellulosic wall,contrary to earlier reports for other species, persists aroundthe microspore mother cells until the late tetrad stage in bothspecies. In C. persicum, it dissolves to release the callose-encasedtetrads, and in A. tuberosum, il dissolves simultaneously withcallose at the time of release of microspores. Allium tuberosum Rottl., Cyclamen persicum Mill., microsporogenesis, microspore mother cell     

Pollen Wall Development of Xiphidium coeruleum (Haemodoraceae) and its Systematic Implications

The development of the pollen grain wall in Xiphidium coeruleum(Haemodoraceae) was studied using TEM and cytochemical stainingtechniques. Microsporocyte ontogeny initiates with the degradationof the cellulosic cell wall and subsequent deposition of a thickcallosic cell wall. Following callose deposition, successivemeiosis occurs, resulting in a tetragonal tetrad of microspores.during meiosis, the cell walls of the tapetum break down, releasingthe syncytial periplasmodium. Irregular non-sporopollenous globularbodies are deposited in this peripheral periplasmodium, whichis rich in ER, golgi bodies, vesicles, and characteristic starchplastids. Within the microspore cytoplasm, vesicles, golgi bodies,and plastids are plentiful during the early tetrad stage. Atthis time the plasma membrane of the microspore develops characteristicevaginations. An extracellular membrane, the ‘white line’,is secreted outside the microspore plasma membrane, followedby callose wall degradation. Bead-like deposits of exine orprimexine are deposited at points along the ‘white line’simultaneously on inner and outer surfaces and opposite theoriginal plasma membrane evaginations. The bead-like exine depositscontinue to grow during the release of the microspores and developinto laterally appressed, rod-shaped ektexinous elements havinga tangentially oriented commissure, the vestige of the original‘white line’. The mature intine is two-layered,the outer exintine containing radially oriented vesicular structures,which are apparently derived from plasma membrane extensions.Exine development in Xiphidium is similar to ‘nexine 1’development in Lilium and may have evolved from an ancestraltectate-columellate condition by the loss of the sexine. Walldevelopment in members of the Zingiberales is strikingly similarto that reported here for the Haemodoraceae—evidence ofa possible relationship between the two taxa. Xiphidium coeruleum, Haemodoraceae, pollen, tapetum, development, exine     

Involvement of Cell Wall-Bound Diferulic Acid in Light-Induced Decrease in Growth Rate and Cell Wall Extensibility of Oryza Coleoptiles

Irradiation of white fluorescent light (5 W m^–2) inhibitedthe growth of Oryza coleoptiles. Light irradiation increasedstress-relaxation parameters of coleoptile cell walls, minimumstressrelaxationtime and relaxation rate, and decreased cellwall extensibility (strain/load). Under light conditions, thecontents of ferulic and diferulic acids ester-linked to thehemicellulosic arabinose residue in cell walls increased andcorrelated with the modification of the cell wall mechanicalproperties. These results suggest that light irradiation enhancesthe formation of diferulic acid bridges in hemicelluloses, makingcell walls mechanically rigid and thus inhibits cell elongationin rice coleoptiles. Also, irrespective of coleoptile age orthe presence of light, the ratio of diferulic acid to ferulicacid was almost constant, suggesting that the rate limitingstep in the formation of diferulic acid bridges in Oryza cellwalls is in the step of feruloylation. (Received September 24, 1991; Accepted December 3, 1991)     

Absence of Callose and Tetrad in the Microsporogenesis of Pandanus odoratissimus with Well-formed Pollen Exine

In the microsporocytes of Pandanus odoratissimus, cytokinesisis successive with centrifugal cleavage in both the meioticdivisions. The dyads move apart from each other after the firstdivision, and the microspores likewise after the second division,so that only monads are formed at the end of meiosis. Althoughno trace of callose wall is found at any stage around the microsporocyteor microspore, fertile, monocolpate pollen with well-developed,spinescent exine develops, and is shed at the two-celled stage. Pandanus odoratissimus, microsporogenesis, centrifugal cleavage, absence of callose, monad formation     

Octad pollen formation in Cymbopetalum (Annonaceae): the binding mechanism

Our recent study of tetrad pollen formation in Annona (Annonaceae) revealed that after meiosis the callose-cellulose envelope forms a special conjugation with individual microspores and the forthcoming callose digestion is incomplete. The undigested part forms a central binder holding the four microspores of the tetrad together. This process causes the microspores to rotate 180 degrees. In this paper we describe pollen formation in another annonaceous genus, Cymbopetalum, in which the pollen is shed in octads, through use of light microscopy, epifluorescence microscopy, and TEM. In Cymbopetalum, two meiocytes, connected by abundant cytomictic channels, are produced in each sporangium. Octad pollen formation in Cymbopetalum is shown to be comparable to the synchronized formation of two connected Annona tetrads, which then integrate into a single octad. Unique features of Annona polyad formation, e.g. special binding between the callose-cellulose envelopes and microspores, incomplete callose digestion, and microspore rotation, also occur in Cymbopetalum. In addition, formation of the Cymbopetalum octad involves development of a cushion-like structure that binds the distal pronexine of all eight microspores, and there is the production of intine protrusions. The evolutionary origin of the callose-cellulose binding mechanism within the family is discussed.     

Tapetum-Specific Expression of the Gene for an Endo-{beta}-1,3-glucanase Causes Male Sterility in Transgenic Tobacco

A cDNA for a pathogenesis-related endo-ß-1,3-glucanaseisolated from soybean, was fused to an anther tapetum-specificpromoter (Osg6B promoter) isolated from rice and the resultingchimeric gene was introduced into tobacco. The Osg6B promoterbecame active in the anther tapetum during formation of tetradsand the tapetal glucanase activity in the transgenic plantscaused in a significant reduction in the number of fertile pollengrains. Most of the pollen grains were aberrant in shape, lackedgerminal apertures and aggregate of the pollen grains. Granulesof ß-1,3-glucan, which have not previously been reported,were often observed to adhere to the surface of the pollen grains.Further observations revealed that the callose wall was almostabsent in the pollen tetrads of transgenic plants. In wild-typeplants, by contrast, the tetrads were surrounded by callosethat was degraded soon after the tetrad stage to release freemicrospores. Thus, the introduced gene for endo-ß-1,3-endoglucanaseunder the control of the Osg6B promoter caused digestion ofthe callose wall at the beginning of the tetrad stage, a timethat was just a little earlier than the time at which endogenousglucanase activity normal appears. These results demonstratethat premature dissolution of the callose wall in pollen tetradscauses male sterility and suggest that the time at which tapetallyproduced glucanase is activate is critical for the normal developmentof microspores. (Received September 29, 1994; Accepted January 30, 1995)     

Studies on microspore development in liliaceous plants II. The behavior of explanted microspores of the lily, Lilium longiflorum

Uninucleate microspores of Lilium longiflorum from differentlengths of buds were explanted under various culture conditions,and their behavior was studied during subsequent cultures. Thenutritional conditions permitting survival of microspores wererelatively simple, but most of the living cells showed cytologicalabnormalities. A typical type of cell division could only beinduced in explanted microspores at the late G₁ phase of thecell cycle. For the normal development of microspores in culture,the microspore environment with respect to moisture was an importantfactor. The rate of the mitotic cycle of the cultured microsporeswas essentially the same as that for microspores grown undergreenhouse conditions. ¹Department of Medicine, Kochi Medical School, Nangoku, Kochi781-51, Japan. (Received March 25, 1980; )     

Formation of Isodityrosine by Peroxidase Isozymes

Fry, S. C. 1987. Formation of isodityrosine by peroxidase isozymes.—J.exp. Bot. 38: 853–862. Tyrosine residues of extensin are oxidatively coupled in vivoto form isodityrosine bridges, whereas treatment of purifiedextensin with H₂O₂+ peroxidase in vitro yields only dityrosine.Two explanations for the correct mode of coupling in vivo weretested. The first, that the pH of the cell wall is lower thanthat (pH 9-0) at which in vitro experiments have been conducted,provided part of the answer since treatment of L-tyrosine withH₂O₂+peroxidase in vitro at pH 37–5 yielded some isodityrosine.The second, that the wall contains other isozymes of peroxidasethan the basic isozyme usually studied in vitro, appeared unlikelybecause several sharply contrasting isozymes yielded similarisodityrosine: dityrosine ratios from L-tyrosine+ H₂O₂ at anygiven pH. The isozymes were also similar in their ability tooxidize tyrosine-dimers further to higher polymers. It is concludedthat the formation of isodityrosine in vivo is dictated by neighbouringwall molecules, possibly ionically-bound pectins, which modifythe local environment of the tyrosine residues of extensin. Key words: Isodityrosine, peroxidase isozymes, extensin     

The Development of Male and Female Regenerants by In Vitro Androgenesis in Dioecious Plant Melandrium album

We examined induced androgenesis in vitro in the dioecious plantMelandrium album and aimed to produce complete plants from culturedimmature microspores. Flow cytometric analysis of nuclear DNAcontent was used to screen ploidy levels in regenerated plantsand to estimate the nuclear genome size in plants differingin sex. Haploid and spontaneous dihaploid (polyhaploid) femalesdominated among androgenic regenerants. Androgenic males occurredsporadically. They were exclusively dihaploid and geneticallysupermales (AAYY). The progenies obtained as a result of thecrosses between supermales and standard females contained onlymales. This is the first report on complete androgenesis inM. album from the microspores carrying the Y chromosome.Copyright1994, 1999 Academic Press Melandrium album (Miller) Garcke, pollen androgenesis, sex, female, male, supermale, flow-cytometry, nuclear genome size     

Reproduction in Seagrasses: Pollen Development in Thalassia hemprichii, Halophila stipulacea and Thalassodendron ciliatum

The general features of pollen morphogenesis in three marinemonocotyledons, Thalassia hemprichii, Halophila stipulacea andThalassodendron ciliatum, are described in this paper. Thalassia disperses spherical trinucleate pollen grains. Inthis genus simultaneous cytokinesis generally produces an isobilateraltetrad of microspores, but linear and T-shaped configurationsalso occur, together with configurations intermediate betweenisobilateral and T-shaped. Partitioning is followed by a phaseof cellular degeneration affecting one or two, never more, membersof the tetrad. Subsequent development of the surviving, functionalmicrospores does not differ essentially from the pattern ofmorphogenesis in terrestrial flowering plants. Halophila disperses strings of four reniform trinucleate pollengrains contained in a mucilaginous moniliform tube. These ariseby successive transverse partitioning of an elongate mothercell and the linear unit so formed is maintained throughoutpollen development. The tetrad tube substance originates inthe tapetal periplasmodium and deposition begins soon aftermeiosis. Thalassodendron disperses filiform trinucleate pollen grains.The characteristic form of the pollen in this genus is attainedduring post-meiotic growth and differentiation, as in othergenera belonging to the same family. This contrasts with thesituation in seagrasses belonging to the Zosteraceae where thefiliform shape is established before meiosis. Precocious divisionof the microspore nucleus in Thalassodendron launches the binucleatepollen phase soon after the spores separate from the tetrad.The division precedes the vacuolate period; again, this is afeature of the family. In Thalassia the tapetal periplasmodium is progressively transformedinto thecal slime. In Thalassodendron and Halophila the periplasmodialresidue forms a superficial coating on the pollen wall and tetradtube. These products could be implicated in attachment and recognitionof the pollen at the stigma surface. Thalassia hemprichii, Halophila stipulacea, Thalassodendron ciliatum, seagrasses, pollen development     

A Comparison of Anther Tissue Development in Male Sterile Aloe vera and Male Fertile Aloe ciliaris

Cytological differences between the anther development of amale sterile and a male fertile Aloe species are used to explaininteractions between anther tissues. Some deviations in thelayers of the locule wall and the microspores of the male sterileanther are related to each other and their biological functionsare discussed. The cytological development of the male sterility,which can be observed shortly after meiosis, seems to be restrictedto the locular cavity. The tapetal development and breakdownare normal, apart from the size of some orbicules. However,the pollenkitt is not transported to the pollengrains, whichstrongly supports our theory that this process is mechanicallypollen-controlled. The development of the epidermal and endothecialcells is normal, except in a part of the anthers where thesecells do not expand, after which dehiscence is incomplete. Thelatter process is discussed in relation to the deviations insidethe locular cavity. Aloe vera (L.) Burm. fil., Aloe ciliaris Haw., Liliaceae, male sterility, tapetum, pollenkitt, endothecium, anther dehiscence     

Nitrate Supply and the Biophysics of Leaf Growth in Salix viminalis

The influence of nitrogen on leaf area development and the biophysicsof leaf growth was studied using clonal plants of the shrubwillow, Salix viminalis grown with either optimal (High N) orsub-optimal (Low N) supplies of nitrate. Leaf growth rate andfinal leaf size were reduced in the sub-optimal treatment andthe data suggest that in young rapidly growing leaves, thiswas primarily due to changes in cell wall properties, sincecell wall extensibility (% plasticity) was reduced in the LowN plants. The biophysical regulation of leaf cell expansion also differedwith nitrogen treatment as leaves aged. In the High N leaves,leaf cell turgor pressure (P) increased with age whilst in theLow N leaves P declined with age, again suggesting that foryoung leaves, cell wall plasticity limited expansion in theLow N plants. Measurements of cell wall properties showed thatcell wall elasticity (%E) was not influenced by nitrogen treatmentand remained constant regardless of leaf age. Key words: Salix, cell wall extensibility, nitrogen nutrition, biophysics of leaf growth     

Cell Expansion and Wall pH in the Fern Regnellidium diphyllum, A Plant Lacking Acidinduced Growth

Petioles of the semi-aquatic fern Regnellidium diphyllum donot show acid growth but low wall pH is a necessary conditionfor maximum rates of IAA-induced cell expansion. Measurementsof wall pH by two indirect methods indicate an unusually lowvalue, in the range pH 4 to 5. This is one to two pH units belowthat estimated for petioles of the semi-aquatic dicotyledonNymphoides peltata, a species in which IAA aand ethylene causegrowth responses very similar to those in Regnellidium but whereacid growth occurs. Having shown previously that fusicoccinenhances proton secretion in both Regnellidium and Nymphoides,we now show that although it causes a reduction in the estimatedapoplast pH to below 4·0 in Regnellidium, cell expansionis not promoted. The FC-induced reduction in pH in Nymphoidesis less and occurs more slowly, but growth is promoted significantly;when IAA and fusicoccin are present together, growth promotionis approximately additive for Nymphoides A model is proposed for Regnellidium in which equilibrium wallpH is maintained at a low value that is optimal for acid growth,the availability of acid-labile sites in the wall being thechief limitation to cell extension. We suggest that this controlmechanism may be widespread for organs without a cuticle, includingroots and the gametophytes of lower plants growing in acidicconditions. Key words: Acid growth, wall pH, fusicoccin, Regnellidium diphyllum, Nymphoides peltata     

Tetrad pollen formation in quartet mutants of Arabidopsis thaliana is associated with persistence of pectic polysaccharides of the pollen mother cell wall

The quartet (qrt) mutants of Arabidopsis thaliana produce tetrad pollen in which microspores fail to separate during pollen development. Because the amount of callose deposition between microspores is correlated with tetrad pollen formation in other species, and because pectin is implicated as playing a role in cell adhesion, these cell-wall components in wild-type and mutant anthers were visualized by immunofluorescence microscopy at different stages of microsporogenesis. In wild-type, callose was detected around the pollen mother cell at the onset of meiosis and around the microspores during the tetrad stage. Microspores were released into the anther locule at the stage where callose was no longer detected. Deposition and degradation of callose during tetrad pollen formation in qrt1 and qrt2 mutants were indistinguishable from those in wild-type. Enzymatic removal of callose from wild-type microspores at the tetrad stage did not release the microspores, suggesting that callose removal is not sufficient to disperse the microspores in wild-type. Pectic components were detected in the primary wall of the pollen mother cell. This wall surrounded the callosic wall around the pollen mother cell and the microspores during the tetrad stage. In wild-type, pectic components of this wall were no longer detectable at the time of microspore release. However, in qrt1 and qrt2 mutants, pectic components of this wall persisted after callose degradation. This result suggests that failure of pectin degradation in the pollen mother cell wall is associated with tetrad pollen formation in qrt mutants, and indicates that QRT1 and QRT2 may be required for cell type-specific pectin degradation to separate microspores.     

Is The Special Callose Wall of Microsporocytes an Impermeable Barrier?

During the course of cytochemical studies in sugar beet anthers(Beta vulgaris L.) to test the applicability of cerium methodsfor the ultrastructural localization of enzymatic activity inplant tissues, new evidence was obtained that questions theimpermeability of the special callose wall surrounding the tetradof microspores. Cerous ions, added to cytochemical media asa potential capture agent for enzymatically-produced hydrogenperoxide, showed binding to cell walls and plasma membranesexclusively in the zone of mechanical injury to the tissues,which may correspond to sites of hydrogen peroxide formationas a consequence of wounding. The cerium perhydroxide precipitateformed as a result of this reaction was localized within thecell walls of anther tissues, inside the callose surroundingtetrads of microspore and in the primexine layer of the microsporewall. The results of this study provide evidence for callosepermeability in in vivo conditions, for at least some substancessuch as cerous ions or cerium perhydroxide. Key words: Callose, cerous ions/cerium perhydroxide, permeability     

Auxin Changes Both the Extensibility and the Yield Threshold of the Cell Wall of Vigna Hypocotyls

Elongation of plant stem is governed by two simultaneous processes:irreversible yielding of the cell wall and uptake of water.Among many candidates for the parameters that regulate and/or restrict growth, we focused on the mechanical propertiesof the cell wall and determined those parameters that governthe process of IAA-induced growth by means of the pressure-jumpmethod combined with the pressure-probe technique. The elongation growth of segments excised from the elongationzone of Vigna hypocotyls was accelerated by xylem perfusionwith 10^–4 M IAA. During the promotion of growth, boththe extensibility () of the cell wall and the effective turgor(Pⁱ–Y) increased while only a little or no change in theintracellular pressure (Pⁱ) occurred. These results indicate that IAA increases not only the extensibilityof the cell wall but also the effective turgor, i.e., the drivingforce for yielding of the cell wall. However, the driving forceis not increased by the increase in Pⁱ but by the decrease inthe yield threshold (Y). These results suggest that Y is adjustableduring the regulation of growth. ¹Present address: Department of Biology, Faculty of Science,Okayama University, Okayama, 700 Japan (Received September 20, 1990; Accepted November 27, 1990)     

The Effect of pH on the Binding of Calcium to Pea Epicotyl Cell Walls and its Implications for the Control of Cell Extension

Cell walls were prepared from the epicotyls of dark-grown pea(Pisum sativum L.) seedlings. The walls were found to bind externally-added⁴⁵Ca²⁺, with a binding constant of 4 ? 10^–4 mol dm^–3and a maximum capacity of 1.5 ? 10^–8 g-ions of Ca²⁺ perg fresh weight of epicotyl. The binding capacity decreased asthe pH of the medium was decreased below 6.0, suggesting thatthe calcium was bound by an anionic group with an apparent pKof 4.7. More than half the calcium binding was due to polygalacturonicacid in the wall, since up to 60% of the calcium binding capacitywas removed by pre-incubation of the cell walls with polygalacturonase(E.C.3.2.1.15). Only small decreases in calcium binding wereseen following pre-incubation with protease, nucleases, phospholipaseand hemicellulase. These results indicate that calcium willbe displaced from the cell wall at hydrogen ion concentrationswhich are known to occur in the wall during wall extension.They are consistent with a mechanism by which calcium inhibitswall extension by forming ionic bridges between polygalacturonicacid molecules, and also with the hypothesis that calcium andhydrogen ions exert opposing influences on cell wall extensionby competing for the same binding sites on the polygalacturonicacid. Key words: Pea epicotyl, Cell wall, Calcium, pH     

Microsporogenesis, Pollination, Pollen Germination and Male Gametophyte Development in Taxus brevifolia

Taxus brevifolia(Nutt.), commonly known as Pacific or westernyew, is a conifer native to the Pacific northwest of North America.Contrary to other Taxus species, T. brevifolia staminate strobiliare usually located on 2-year-old foliage although they mayoccur on foliage from 1 to 5-years-old. This delayed staminatestrobilus development may be an adaptation to the low lightenvironment where T. brevifolia grows. Microsporogenesis occurredin the autumn preceding pollination. Successive divisions producedisobilateral tetrads visible as early as mid-October. Over-winteringstaminate strobili usually contained separate microspores. In1996 to 1999, pollination occurred in March and April in twonatural forest sites on southern Vancouver Island, British Columbia,Canada. The low amounts of airborne pollen and prolonged pollinationperiod indicated low pollination success within T. brevifolia.Female receptivity was measured by the presence of a pollinationdrop. Protandry up to 18 d was observed. In vitro pollen germinationwas moderate to good, ranging from 65 to 88% depending on thetree and year. DAPI fluorescence staining showed successfulmale gametophyte development in vitro. The microspore dividedforming a tube nucleus and generative cell within 3 d of culture.The generative cell then divided forming a sterile nucleus andspermatogenous nucleus after 17 d. The spermatogenous nucleusacquired a cell wall then divided forming two equal sperm after24 d. Copyright 2000 Annals of Botany Company Taxus brevifolia, Pacific yew, microsporogenesis, pollination, pollen germination, male gametophyte development