Role of a callose synthase zymogen in regulating wall deposition in pollen tubes of Nicotiana alata Link et Otto

The callose synthase (CalS) activity of membrane preparations from cultured Nicotiana alata Link & Otto pollen tubes is increased several-fold by treatment with trypsin in the presence of digitonin, possibly due to activation of an inactive (zymogen) form of the enzyme. Active and inactive forms of CalS are also present in stylar-grown tubes. Callose deposition was first detected immediately after germination of pollen grains in liquid medium, at the rim of the germination aperture. During tube growth the 3-linked glucan backbone of callose was deposited at an increasing rate, reaching a maximum of 65 mg h⁻¹ in tubes grown from 1 g pollen. Callose synthase activity was first detected immediately after germination, and then also increased substantially during tube growth. Trypsin caused activation of CalS throughout a 30-h time course of tube growth, but the degree of activation was higher for younger pollen tubes. Over a 10-fold range of callose deposition rates, the assayed CalS activity was sufficient to account for the rate of callose deposition without trypsin activation, implying that the form of CalS active in isolated membranes is responsible for callose deposition in intact pollen tubes. Sucrose-density-gradient centrifugation separated a lighter, intracellular membrane fraction containing only inactive CalS from a heavier, plasma-membrane fraction containing both active and inactive CalS, with younger pollen tubes containing relatively more of the inactive intracellular enzyme. The increasing rate of callose deposition during pollen-tube growth may thus be caused by the transport of inactive forms of CalS from intracellular membranes to the plasma membrane, followed by the regulated activation of these inactive forms in this final location. Received: 1 December 1998 / Accepted: 21 January 1999     

A Fluorescent Brightener used for Pollen Tube Identification In Vivo

Squash preparations of styles stained in watersoluble aniline blue and viewed under ultra-violet illumination are regularly used for examining pollen tubes because the callose plugs fluoresce brightly under these conditions. Tubes are therefore clearly distinguish from the astylar tissue and may be readily counted and measured. This method has proved to be quite unsatisfactory for plum pollen tubes, since they contain very few cause plugs and better results have been obtained with a mixed stain of 0.1% aniline blue and 0.07% of the fluorescent brightener 'Calcofluor White M2R New'. Styles are softened by autoclaving in 50 g/1 sodium sulphite, rinsed and stained for ten minutes, then squashed and examined with a fluorescence microscope in the usual way. Callose deposits, when present, fluoresce bright yellow, but lengths of tube with no deposits can also be clearly identified and followed, permitting easier, faster and more accurate assessments of pollen tube length and numbers in plum and pear styles.     

Rhizobium-stimulated Callose Formation in Clover Root Hairs and its Relation to Infection

Callose was detected in the cell walls of the tips of growingroot hairs of Trifolium species and the non-legume Phleum pratenseusing u.v. fluorescence of fresh material stained with 0·005%aniline blue. Inoculation of the roots with Rhizobium trifolii,R. leguminosarum, R. meliloti, and R. japonicum, or additionof 10^–7 and 10^–8 M indole-3-acetic acid (IAA) increasedtip callose formation. Most tip callose was formed at 12 °C, and amounts declinedprogressively at 18, 24, and 30 °C, with very little formedat 36 °C. Tip calloso usually became less and disappearedin individual root hairs as they aged. Callose which appeared prominently in the host cell walls atthe points of initiation of infection threads did not usuallydisappear as the hairs matured. There was little or no extensionof callose along the infection thread and none in the threadtip or in the cell nucleus. Presumptive regions of callose hadsimilar structure and electron density as root hair wall materialand were sometimes related to arrays of vesicles in the hostcytoplasm. The external surface of the hair wall bore smallpegs or papillae (0·1–0·2 µm) continuouswith the outer layer of the wall and possibly associated withattachment of bacteria. Bacteria were usually umboriate at thepoint of attachment and their polyphosphate granules were muchlarger near the root hair than at the distal end.     

Location of cellulose and callose in pollen tubes and grains of Nicotiana tabacum

The distribution of cellulose and callose in the walls of pollen tubes and grains of Nicotiana tabacum L. was examined by electron microscopy using gold-labelled cellobiohydrolase for cellulose and a (1,3)-β-D-glucan-specific monoclonal antibody for callose. These probes provided the first direct evidence that cellulose co-locates with callose in the inner, electron-lucent layer of the pollen-tube wall, while both polymers are absent from the outer, fibrillar layer. Neither cellulose nor callose are present in the wall at the pollen-tube tip or in cytoplasmic vesicles. Cellulose is first detected approximately 5–15 μm behind the growing tube tip, just before a visible inner wall layer commences, whereas callose is first observed in the inner wall layer approximately 30 μm behind the tip. Callose was present throughout transverse plugs, whereas cellulose was most abundant towards the outer regions of these plugs. This same distribution of cellulose and callose was also observed in pollen-tube walls of N. alata Link et Otto, Brassica campestris L. and Lilium longiflorum Thunb. In pollen grains of N. tabacum, cellulose is present in the intine layer of the wall throughout germination, but no callose is present. Callose appears in grains by 4 h after germination, increasing in amount over at least the first 18 h, and is located at the interface between the intine and the plasma membrane. This differential distribution of cellulose and callose in both pollen tubes and grains has implications for the nature of the β-glucan biosynthetic machinery. Received: 20 February 1988 / Accepted: 25 March 1998     

The division of the generative nucleus and the formation of callose plugs in pollen tubes of Aechmea fasciata (Bromeliaceae) cultured in vitro

The effect of different external factors on pollen germination and pollen tube growth is well documented for several species. On the other hand the consequences of these factors on the division of the generative nucleus and the formation of callose plugs are less known. In this study we report the effect of medium pH, 2-[N-morpholino]ethanesulfonic acid (MES) buffer, sucrose concentration, partial substitution of sucrose by polyethyleneglycol (PEG) 6000, arginine (Arg), and pollen density on the following parameters: pollen germination, pollen tube length, division of the generative nucleus, and the formation of callose plugs. We also studied the different developmental processes in relation to time. The optimal pH for all parameters tested was 6.7. In particular, the division of the generative nucleus and callose plug deposition were inhibited at lower pH values. MES buffer had a toxic effect; both pollen germination and pollen tube length were lowered. MES buffer also influenced migration of the male germ unit (MGU), the second mitotic division, and the formation of callose plugs. A sucrose concentration of 10% was optimal for pollen germination, pollen tube growth rate and final pollen tube length, as well as for division of the generative nucleus and the production of callose plugs. Partial substitution of sucrose by PEG 6000 had no influence on pollen germination and pollen tube length. However, in these pollen tubes the MGU often did not migrate and no callose plugs were observed. Pollen tube growth was independent of the migration of the MGU and the deposition of callose plugs. In previous experiments Arg proved to be positive for the division of the generative nucleus in pollen tubes cultured in vitro. Here, we found that more pollen tubes had callose plugs and more callose plugs per pollen tube were produced on medium with Arg. After the MGU migrated into the pollen tube (1 h after cultivation), callose plugs were deposited (3 h). After 8 h the first sperm cells were produced. The MGU moved away from the active pollen tube tip until the second pollen mitosis occurred, thereafter the distance from the MGU to the pollen tube tip diminished. Callose plug deposition never started prior to MGU migration into the pollen tube. Pollen tubes without a MGU also lack callose plugs (±30% of the total number of pollen tubes). Furthermore, we found a correlation between the occurrence of sperm cells in pollen tubes and the synthesis of callose plugs.     

Callose (β-1,3 glucan) is essential for <Emphasis Type="Italic">Arabidopsis</Emphasis>pollen wall patterning,but not tube growth

Background  

Callose (β-1,3 glucan) separates developing pollen grains, preventing their underlying walls (exine) from fusing. The pollen tubes that transport sperm to female gametes also contain callose, both in their walls as well as in the plugs that segment growing tubes. Mutations in CalS5, one of several Arabidopsis β-1,3 glucan synthases, were previously shown to disrupt callose formation around developing microspores, causing aberrations in exine patterning, degeneration of developing microspores, and pollen sterility.     

β-Glucan synthetase II activity upon callose formation in the flower ofArabidopsis thaliana

Callose formation was observed in the pollens during flower development and pollen tube grown in the pistil ofA. thaliana. The accumulation of callose occurred in the tetrad in the flower bud and pollen tube. Therefore, the activity of β-glucan synthetase II (GS II), which is responsible for synthesizing the callose, was measured in the flowers on the same developmental stages. The enzyme activity was increased by about 10% while the level of callose contents was increased by about 70% in tetrads. Then, callose accumulation was increased during pollen tube growth by about 30% higher than the other stages and enzyme activity was detected, 30% more too. These results suggest that callose plays an important role in the growth of pollen and pollen tube by increasing GS II activity.     

Specificity of the Callose Response in Stigmas of Brassica

The specificity of pollen in the induction of callose in stigmasof Brassica spp. has been examined. Callose is induced by fractionsfrom self pollen and pollen of another crucifer, Sinapis arvensis,but not from compatible pollen. Response is blocked by pre-treatment of stigmas with concanavalinA and Triton X-100. Brassica spp., callose, incompatibility, pollen-stigma interactions, recognition     

Pollen germinates precociously in the anthers of raring-to-go, an Arabidopsis gametophytic mutant

Pollen hydration is usually tightly regulated and occurs in vivo only when desiccated pollen grains acquire water from the female, thus enabling pollen tube growth. Pollen tubes are easily visualized by staining with decolorized aniline blue, a stain specific for callose. We identified a mutant, raring-to-go, in which pollen grains stained for callose before anther dehiscence. When raring-to-go plants are transferred to high humidity, pollen tubes dramatically elongate within the anther. As early as the bicellular stage, affected pollen grains in raring-to-go plants acquire or retain water within the anther, and precociously germinate. Thus, the requirement for contact with the female is circumvented. We used pollen tetrad analysis to show that raring-to-go is a gametophytic mutation, to our knowledge the first gametophytic mutation in Arabidopsis that affects early events in the pollination pathway. To aid in identifying raring-to-go alleles, we devised a new technique for screening pollen in bulk with decolorized aniline blue. We screened a new M(1) mutagenized population and identified several additional mutants with a raring-to-go-like phenotype, demonstrating the usefulness of this technique. Further, we isolated other mutants (gift-wrapped pollen, polka dot pollen, and emotionally fragile pollen) with unexpected patterns of callose staining. We suggest that raring-to-go and these other mutants may help dissect components of the pathway that regulates pollen hydration and pollen tube growth.     

Loss of callose in the stigma papillae does not affect the Brassica self-incompatibility phenotype

As part of the Brassicaceae self-incompatibility response, callose is deposited in the stigma papillar cells. To determine if callose plays an important role in the rejection of incompatible pollen by the stigma, transgenic Brassica napus. L. plants were produced which express the tobacco β-1,3-glucanase cDNA (the enzyme which degrades callose) in the stigma papillae. Using aniline blue fluorescence, little or no callose was detected in the papillar cells of transgenic stigmas. However, the self-incompatibility system appeared to be unaffected based on the lack of pollen tube growth and the subsequent lack of seed set. The transgene had no effect on compatible pollinations. Thus, while callose deposition is associated with the B. napus self-incompatibility response, it is not required for the rejection of incompatible pollen. Received: 14 March 1997 / Accepted: 15 April 1997     

Applications of fluorochromes to pollen biology. II. The DNA probes ethidium bromide and Hoechst 33258 in conjunction with the callose-specific aniline blue fluorochrome

Techniques are described for detection of pollen grain and pollen tube nuclei using the fluorescent DNA probes ethidium bromide or Hoechst 33258, in conjunction with the aniline blue fluorochrome sirofluor, which stains the callose component of pollen tube walls and plugs. The DNA probes, which may be used either as vital stains or following fixation, permit discrimination between vegetative and generative or sperm nuclei. Double staining with sirofluor allows location of nuclei within pollen tubes grown in vitro, and when used after pollination enables the viewer to discriminate between nuclei within the pollen tube vs. nuclei of the pistil tissue.     

Lactic Acid Clearing and Fluorescent Staining for Demonstration of Sieve Tubes

Sieve tubes of the phloem in cleared plant parts can be located by means of a staining reaction specific for callose. The plant part is decolourized in 1:3 glacial acetic acid-95% ethanol and cleared in hot 85% lactic acid at 98-100 C. Callose is not dissolved by this treatment and is then stained with 0.01% analine blue in 0.07 M phosphate buffer, pH 7.5, and observed by fluorescence microscopy. A sieve tube is recognized by the bright yellow fluorescence of the callose on its sieve plates. In most tissues, a natural light yellow fluorescence of the parenchyma cells is evident after the clearing step. This intensifies upon staining with analine blue and tends to make the tissue opaque, but it can be minimized by quick-killing of the tissue before commencing the decolourization. The procedure gives best results when applied to young tissues in which interference from the natural yellow fluorescence of lignified cells such as xylem elements and phloem fibers is minimal. Callose plugs in pollen tubes were also shown in intact, cleared styles.     

Late-acting self-incompatibility in tea plant (<Emphasis Type="Italic">Camellia sinensis</Emphasis>)

The self-incompatibility of tea plant (Camellia sinensis (L.) O. Kuntze) was studied with the methods of aniline blue fluorescence assay and paraffin sections. The characteristics of pollen tube elongation after hand pollination was analyzed in 4 tea cultivars, including ‘Keemenzhong’, ‘Longjing-changye’, ‘Fuding-dabaicha’ and ‘Yabukita’, under self-pollination and cross-pollination, respectively. Although there were some difference among cultivars, pollen tubes elongated through the style and reach the ovary successfully at 48 h after pollination for both cross- and self-pollen tubes in all the four cultivars of tea. Pollen tubes entered into the ovule micropyles, however, only for cross-pollination, but not for self-pollination. Pollen tubes of selfing plants, failed in fertilizing, seemed have some difficulties to enter the ovule. All of which indicated that the self-incompatibility of tea plant is a late-acting self-incompatibility system (LSI) or an ovarian sterility (OS), in which the self incompatibility was due to none self pollen tube penetrating into the ovule and no fertilization.     

Applications of Fluorochjromes to Pollen Biology. Ii. The Dna Probes Ethidium Bromide and Hoechst 33258 in Conjunction with the Callose-Specific Aniline Blue Fluorochrome

Techniques are described for detection of pollen grain and pollen tube nuclei using the fluorescent DNA probes ethidium bromide or Hoechst 33258, in conjunction with the aniline blue fluorochrome sirofluor, which stains the callose component of pollen tube walls and plugs. The DNA probes, which may be used either as vital stains or following fixation, permit discrimination between vegetative and generative or sperm nuclei. Double staining with sirofluor allows location of nuclei within pollen tubes grown in vitro, and when used after pollination enables the viewer to discriminate between nuclei within the pollen tube vs. nuclei of the pistil tissue.     

Immunocytochemical localization of callose in the germinated pollen ofCamellia japonica

Summary A polyclonal antibody against -1,3-glucan, callose, extracted from the pollen tube wall ofCamellia japonica was raised in mice and, using it as a probe, the localization of callose in the germinated pollen was studied. By confocal laser scanning microscopy, callose was found in the tip region of the pollen tube and the tube wall; the immuno-fluorescence in the tube wall was less toward the base of the tube. In contrast, the tip region did not fluoresce although the whole of the tube wall did strongly with aniline blue, the specific dye for callose. Immuno-electron microscopy showed that callose was also found in Golgi vesicles which concentrated in the tip region of the pollen tube, the inner layer of the tube wall, callose plugs, and Golgi vesicles in the pollen grain. Immuno-gold labeling was often detected on the fibrous structures in Golgi vesicles and callose plugs. Based on these results, the participation of Golgi vesicles in the formation of the tube wall and callose plugs was discussed.Abbreviation TBS Tris-buffered saline - Tris Tris(hydroxy-methyl)-aminomethane - PBS phosphate-buffered saline - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - CLSM confocal laser scanning microscopy - DP degree of polymerization     

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     

INCOMPATIBILITY IN AMSINCKIA GRANDIFLORA (BORAGINACEAE): DISTRIBUTION OF CALLOSE PLUGS AND POLLEN TUBES FOLLOWING INTER- AND INTRAMORPH CROSSES

The distribution of callose plugs and pollen tubes was investigated following inter- and intramorph crosses of Amsinckia grandiflora (Boraginaceae), a distylous species possessing cryptic self-incompatibility. Callose plug distribution provided a good indication of the distribution of pollen tubes. Compared to intramorph crosses, many more callose plugs and pollen tubes were found in basal stylar regions following intermorph crosses, indicating that differential pollen tube growth is a likely cause of cryptic self-incompatibility. The incompatibility response differed for the floral morphs: in the pin (long-styled) morph pollen tubes were most likely to cease growth in the midstylar region, while inhibition was more likely to occur in the upper stylar region of the thrum (short-styled) morph. There was no evidence of stigmatic inhibition of pollen tubes for either morph, although the incompatibility response in the Boraginaceae is normally located in the stigmatic region.     

Callose Deposition During Megagametogenesis in Two Species of Oenothera

Callose deposits are present both in degenerating megasporesof the heteropolar tetrad in Oenothera hookeri and in degeneratingembryo sacs of the homopolar developing tetrad in O biennis.They are partially continuous with the cell wall and partiallyenclosed in the degenerating cytoplasm and show electron opaquebands within a less electron opaque material Vesicles calledcallose grains are present in the degenerating cytoplasm ofthe embryo sac in O biennis These show an electron opaque fibnllaror granular core surrounded by a halo of low electron opacity Similarities in fine structure between callose deposits of femalegametophytes which follow the degenerating pathway of development,and callose plugs present in pollen tubes during their growth,are discussed. Oenothera, evening primrose, megagametogenesis, megasporogenesis, callose, ultrastructure     

Self-incompatibility of Zinnia angustifolia HBK (compositae)

Summary Visible light and UV epifluorescence microscopy were used to assess self-incompatibility (SI) in Zinnia angustifolia clones. Pistils were fixed 24 h after pollination and stained either with aniline blue in lactophenol (visible light microscopy) or decolorized aniline blue (fluorescence microscopy). Percentage of florets with embryos 21 days following pollination (% embryo set) was used as a control. Embryo set following self- or incompatible cross-pollinations ranged from 0% to 9.9%, whereas compatible crosses yielded 55.5%–87.1% embryo set. Observations using visible light microscopy indicated pollen load and number of germinated grains were significantly higher for compatible compared to incompatible crosses, and both variables were positively correlated (r = 0.89–0.96) to % embryo set. Examinations with UV epifluorescence microscopy revealed pollen load was higher and little or no callose accumulated in stigmatic papillae following compatible crosses, whereas for incompatible crosses, pollen load was low and callose lenticules were deposited in stigmatic papillae; the correlation between pollen load and % embryo set was 0.89. The intensity of callose fluorescence of the pollen tube-papillae attachment sites was quantitatively measured via micro spectrofluorometry. Callose fluorescence intensity ranged from 47.9% to 62.6% for incompatible and from 6.4% to 9.9% for compatible crosses, and was negatively correlated (r= — 0.95) with % embryo set. Microscopal techniques permit rapid assessment of SI and may be used routinely when each observed or measured parameter is highly correlated to the incompatibility response.Publication no. 2898 of the Massachusetts Agricultural Experiment Station.