Glycoproteins 50, 55, 59 and 64 kDa with affinity to the lectin ConA occurring abundantly in mature tobacco pollen were shown to exhibit high tolerance against heating at 90°C for 30°min. The 59 kDa glycoprotein (GP59) was isolated by affinity chromatography on ConA-agarose followed by 2D-electrophoresis and identified by MS analysis as tobacco calreticulin with approximate pI 4.2. Identification of the protein was confirmed by immunoblotting with human anti-calreticulin and by labelling with a specific dye for Ca2+ -binding proteins (Stains All). Two acidic isoforms of 50 kDa glycoprotein in addition to GP59 displayed homology to calreticulin. RT-PCR revealed the presence of transcripts for calreticulin 59 kDa at all the stages of pollen development from microspore mitosis through the first 24 h of pollen tube growth. Immunodetection with anti-calreticulin and affinity to ConA on Western blots of total soluble proteins separated by 1D-SDS-PAGE showed that the protein first occurred at the mid-bicellular pollen stage, accumulated during pollen maturation and disappeared during 24 h of pollen tube growth. A thermotolerant form of GP59 was detected only in the terminal phase of pollen maturation and during 8 h of pollen tube growth. Results indicated that calreticulin 59 kDa is transcribed, translated and undergoes post-translational modification at a number of different stages of pollen development and that thermotolerance of the protein in mature pollen may be associated with high glycosylation. The thermotolerance of these glycoproteins could play a role in the protection of pollen against stress factors during dehydration and dispersal.

Tobacco (Nicotiana tabacum L.) microspores at the time of mitosis are characterized by the abundant occurrence of 92- and 98-kDa glycoproteins (GP92 and GP98). GP92 is a soluble protein while GP98 is bound to the insoluble microspore fraction. Both glycoproteins were isolated by affinity chromatography and SDS-PAGE and analysed by MS. Peptide sequences were determined by mu-HPLC/nano-ESI-MS/MS (electrospray ionization tandem MS). GP92 displayed homology to beta-galactosidase (EC 3.2.1.23) and GP98 to beta-xylosidase (EC 3.2.1.37) from Arabidopsis thaliana (L.) Heynh. The activities of the two enzymes in microspore and pollen extracts of tobacco exhibited similar developmental changes to the occurrence of GP92 and GP98, with a maximum around microspore mitosis. These two glycoproteins are the first identified enzymes characteristic of mitotic microspores. Arabidopsis transcriptomic data for five beta-galactosidase and three beta-xylosidase genes abundantly expressed in pollen were verified by reverse transcription-PCR of RNA from different stages of Arabidopsis pollen development and from various parts of the sporophyte. The results showed abundant expression of two genes (At5g20710, At1g31740) homologous to tobacco GP92 in microspores and early pollen, and of three genes (At5g56870, At2g16730 and At4g35010) in maturing pollen. Analysis of beta-xylosidases showed abundant expression of a late pollen-specific gene At3g62710 and low expression of an early gene At5g10560. It is suggested that the early beta-galactosidase and beta-xylosidase genes may participate in cell wall loosening associated with pollen expansion after microspore mitosis and that the products of the late genes may play a role in cell expansion during pollen germination.

Oscillatory growth of pollen tubes has been correlated with oscillatory influxes of the cations Ca(2+), H(+), and K(+). Using an ion-specific vibrating probe, a new circuit was identified that involves oscillatory efflux of the anion Cl(-) at the apex and steady influx along the tube starting at 12 microm distal to the tip. This spatial coupling of influx and efflux sites predicts that a vectorial flux of Cl(-) ion traverses the apical region. The Cl(-) channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid completely inhibited tobacco pollen tube growth at 80 and 20 microM, respectively. Cl(-) channel blockers also induced increases in apical cell volume. The apical 50 micro m of untreated pollen tubes had a mean cell volume of 3905 +/- 75 microm(3). DIDS at 80 microM caused a rapid and lethal cell volume increase to 6206 +/- 171 microm(3), which is at the point of cell bursting at the apex. DIDS was further demonstrated to disrupt Cl(-) efflux from the apex, indicating that Cl(-) flux correlates with pollen tube growth and cell volume status. The signal encoded by inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P(4)] antagonized pollen tube growth, induced cell volume increases, and disrupted Cl(-) efflux. Ins(3,4,5,6)P(4) decreased the mean growth rate by 85%, increased the cell volume to 5997 +/- 148 microm(3), and disrupted normal Cl(-) efflux oscillations. These effects were specific for Ins(3,4,5,6)P(4) and were not mimicked by either Ins(1,3,4,5)P(4) or Ins(1,3,4,5,6)P(5). Growth correlation analysis demonstrated that cycles of Cl(-) efflux were coupled to and temporally in phase with cycles of growth. A role for Cl(-) flux in the dynamic cellular events during growth is assessed. Differential interference contrast microscopy and kymographic analysis of individual growth cycles revealed that vesicles can advance transiently to within 2 to 4 microm of the apex during the phase of maximally increasing Cl(-) efflux, which temporally overlaps the phase of cell elongation during the growth cycle. In summary, these investigations indicate that Cl(-) ion dynamics are an important component in the network of events that regulate pollen tube homeostasis and growth.

The complex cellular events that occur during development of the male gametophyte of higher plants suggest a role for the cytoskeleton. This investigation has revealed that unique microtubule arrays mediate events that occur during microspore development; both actin and microtubule arrays have important roles during the asymmetrical microspore mitosis and unique actin arrays mediate events that occur during early pollen development, Migration of the nucleus to the generative pole during cellular polarization of the microspore is mediated by a microtubule cage that encloses the nucleus. Nuclear position at the generative pole is maintained by an actin net that tethers it to the pole prior to the asymmetrical mitosis. During entry into mitosis, the microtubule cage becomes modified and transforms into the asymmetrical mitotic spindle, Actin is localized within the region of the mitotic spindle and in the phragmoplast, following mitosis, actin networks enclose first the generative cell and then the vegetative nucleus. These actin networks function during migration of the generative cell and vegetative nucleus toward the centre of the pollen grain. Mature pollen contains a dense cortical actin meshwork and a disc-shaped microtubule array enclosing the generative cell. The functional importance of the unique actin and microtubule arrays is verified by their targeted disruption with specific cytoskeletal inhibitors, which disrupt normal development and cellular morphology. In summary, these data provide evidence that the co-ordinated reorganization of unique actin and microtubule arrays is an essential determinant of microspore and pollen development.

The effect of media composition on microspore culture was investigated in one tetraploid and two diploid potatoes. The viability of microspores isolated from 4.5 to 5 mm buds was in the range of 33 to 52%. In media for anther culture, microspores showed no further development and lost viability within 2 days. In M1 medium containing mineral components, sucrose, uridine, cytidine, myo-inositol, glutamine and lactalbumin hydrolysate, 18 to 37% of microspores underwent mitosis within 14 days. Up to 95% of the divisions were symmetric and produced equal nuclei. Some symmetrically divided microspores eventually produced structures with 3 to 10 nuclei. The proportion of the total microspore population producing multinuclear structures reached 9% in diploid clones responsive to anther culture and 1 to 2% in recalcitrant cv. Borka. Symmetric mitoses in M1 medium were induced in the presence of glutamine and lactalbumin hydrolysate. Nucleosides and myo-inositol had no effect on microspore division. In the absence of all organic components except sucrose, most mitoses were asymmetric, formation of multinuclear structures was reduced and most pollen accumulated starch indicative of gametophytic fate. In complete M1 medium, starch accumulation was suppressed. Suppression also occurred in asymmetrically divided microspores, indicating a direct inhibition of pollen development independent of the mode of microspore division. This inhibitory effect of M1 medium might present a stress which triggers the induction of symmetric microspore division and subsequent formation of multinuclear structures.

Anthers of diploid genotypes of Solanum tuberosum capable of androgenesis were cultured on different media to examine the effect on induction of pollen embryogenesis of 2,4-D and lactose. Anthers cultured in callogenic medium with 2,4-D and sucrose produced pollen derived embryoids only exceptionally. When sucrose was replaced by lactose the frequency of embryogenesis was as high or higher than in embryogenic auxin-free medium. Substitution of lactose for sucrose in the embryogenic medium had no effect. Supplementing the embryogenic medium with 2,4-D strongly reduced the frequency of pollen embryoids in the presence of sucrose but not with lactose.

Regulation of expression of a 69-kDa glycoprotein which occurs abundantly in tobacco (Nicotiana tabacum L.) pollen tubes but is absent in ungerminated pollen has been studied in vitro by means of a coupled translation/glycosylation system with RNA isolated from various stages of pollen development. Pollen mRNA could be translated in a rabbit reticulocyte lysate and the products glycosylated with canine pancreatic microsomal membranes. The electrophoretic pattern of translation products obtained with pollen-tube RNA showed a prominent polypeptide with an apparent molecular mass of 58 kDa. In the presence of the canine pancreatic microsomal membranes this polypeptide was glycosylated, producing the 69-kDa glycoprotein. The presence of mRNA encoding the 58-kDa precursor polypeptide was also demonstrated in ungerminated pollen and in young mid-binucleate pollen isolated from anthers. Initiation of synthesis of the 69-kDa glycoprotein at the onset of pollen germination thus occurs through unmasking of the mRNA transcribed during pollen differentiation and stored during pollen maturation and dormancy in an inactive state.

In the cycle of sexual plant reproduction consisting in alternation of diploid and haploid generations, the diploid sporophyte produces spores, the mitotic division of which gives rise to the haploid gamete-producing gametophyte. According to this general scheme, the microsporocytes in the anther undergo meiosis to produce microspores through the process of microsporogenesis. The mitotic division of the unicellular microspore starts the ontogeny of pollen (microgametophyte) during which two gametes are produced by a process of gametogenesis. The pollen grain, the developmental equivalent of the embryo sac (macrogametophyte) should thus not be termed microspore, similarly as the embryo sac is not called macrospore, and the term microgametophyte is to be limited to the two- or three-cellular structures arising from microspore. Using this phylogeny–based terminology, the two postmeiotic mitotic divisions should be termed microsporemitosis and pollenmitosis, rather than 1st and 2nd pollen mitosis.

The potential of immature pollen to deviate from its normal developmental pathway, giving rise to haploid embryos and plants, directly and asexually, has been known for a long time and is used in plant breeding for dihaploid production through another culture (Heberle-Bors, 1989, and references therein). For basic and applied research, it would be very interesting to find out the molecular mechanisms underlying the transformation of the male gametophyte into an embryogenic cell. Another culture, however, is not a suitable experimental system for these studies. The anther contains a heterogeneous cell population (a small percentage of embryogenic pollen, from which the embryos originate, together with normal gametophytic pollen and sporophytic cells from the anther tissues) which does not allow the use of biochemical and molecular techniques to study the process of induction of pollen embryogenesis. In addition, the anther wall can contribute substances, both stimulatory and inhibitory, affecting pollen embryo development in a way difficult to control

Nuclease activity in maturing tobacco anthers is mainly confined to the extracellular fraction and is the lowest in pollen extract. The extracellular fraction further contains phosphodiesterase (exonuclease), 3'- and 5'-nucleotidase and phosphatase activities. The level of enzyme activities exhibits different changes during anther development from the stage of microspore to the maturity. An overall rise occurring after completion of microspore division is followed by a fall of phosphodiesterase, a relative constancy in nucleotidase and a continuous rise of nuclease activities till the stage of almost mature anthers. The nuclease is similar in electrophoretic pattern and in substrate specificity to the earlier described extracellular pollen nuclease classified as plant nuclease I (E.C. 3.1.30.x). The diffusate of mature pollen exhibits as much as 28 % of nuclease activity but less than 4 % of the other phosphohydrolytic activities, with respect to their highest levels in the extracellular anther fraction. Polynucleotide molecules present in this fraction are heterogeneous and of low molecular mass, and the RNA component has high G and low U content, corresponding to the preference of nuclease for poly(U) sequences and to its inability to hydrolyze poly(dG) homopolymers. High nuclease and nucleotidase activities in the extracellular anther fraction coincide with the earlier observed high RNA synthesis and an increase of uptake capacity for uridine in developing pollen. The function of extracellular anther phosphohydrolases is discussed in relation to anther senescence and to pollen maturation.

Mature pollen grains of all 15 species examined release nuclease within the first minutes of contact with a liquid medium. These species include plants with both binucleate and trinucleate pollen and with biotic and abiotic modes of pollination. The enzyme of Pinus nigra pollen exhibits similar characteristics as the earlier described enzyme of tobacco pollen classified as plant nuclease I (E.C. 3.1.30.x). It is shown to be a sugar-unspecific endonuclease with maximal activity at acidic pH and preference for single-stranded molecules. The enzyme produces oligonucleotides and 5'-mononucleotides. Nuclease of the other pollen species also has more or less pronounced preference for denatured as opposed to native DNA. A pH in the range of 5.0-5.9 and 6.8-7.0 is optimal for the enzyme of dicotyledons and cereal species, respectively. Different pollen species release different amounts of nuclease with different specific activities. The enzyme also exhibits species-related variation in electrophoretic mobility and in the number of molecular forms. Molecular sieving of pine pollen diffusate produced one nuclease peak with an apparent molecular mass of 29.5 x 103.