Characterization of oleosins in the pollen coat of Brassica oleracea.

Mature Brassica oleracea pollen grains are covered with a lipophilic pollen coat containing a variety of proteins. Screening of an anther cDNA expression library for the coding sequences of such proteins resulted in the isolation of a number of cDNA clones encoding glycine-rich oleosins. The proteins were shown to be attached to the lipophilic coat material only and to be absent elsewhere in the plant. Within the coat, several forms of the pollen coat oleosin with different molecular weights were detected. The forms are encoded by different transcripts that originate from a single gene. Expression of this gene is restricted to the tapetum and is quantitatively regulated by the water content of the anther. Similar oleosins were found in the pollen coat of B. alboglobra and B. napus.

Expression in anthers of two genes encoding Brassica oleracea transmembrane channel proteins.

Screening of an anther cDNA expression library resulted in the isolation of two almost identical cDNA clones, termed mipA and mipB, showing homology with sequences encoding transmembrane channel proteins from the MIP family. Both clones were expressed in several tissues, but not in pollen. MipA was preferentially expressed in the surrounding sporophytic tissues of stamens. Anthers subjected to drought were induced to accumulate even more mip transcripts, which was entirely due to higher mipA gene expression. On basis of isolation procedures, sequence homology and drought inducibility of mipA we conclude that the encoded proteins probably are constituents of the pollen coat and are aquaporins.

Expression of an isoflavone reductase-like gene enhanced by pollen tube growth in pistils of Solanum tuberosum.

Successful sexual reproduction relies on gene products delivered by the pistil to create an environment suitable for pollen tube growth. These compounds are either produced before pollination or formed during the interactions between pistil and pollen tubes. Here we describe the pollination-enhanced expression of the cp100 gene in pistils of Solanum tuberosum. Temporal analysis of gene expression revealed an enhanced expression already one hour after pollination and lasts more than 72 h. Increase in expression also occurred after touching the stigma and was not restricted to the site of touch but spread into the style. The predicted CP100 protein shows similarity to leguminous isoflavone reductases (IFRs), but belongs to a family of IFR-like NAD(P)H-dependent oxidoreductases present in various plant species.

Role of N-glycosylation of 66 and 69 kDa glycoproteins in wall formation during pollen tube growth in vitro.

Two abundant cell wall glycoproteins (66 and 69 kDa) accumulate during growth in pollen tubes of tobacco. Glycosylation of the proteins was experimentally modified by application of the specific inhibitors tunicamycin and castanospermine to in vitro cultured pollen. Newly synthesized proteins were labeled with a 14C-amino acid mixture supplied to the medium. Modified glycoproteins were extracted from pollen tubes and isolated cell walls, and separated by 1-D and 2-D electrophoresis. The size of the molecules was reduced by tunicamycin and increased by castanospermine, effects which were measurable from the beginning of cultivation. The modification of the glycan moiety did not affect deposition of the proteins in the wall. Cultivation in the continuous presence of either inhibitor led to reduced callose deposition in the secondary cell wall and to inhibition of pollen tube growth. The results suggest that the two proteins play a role in the formation of the callose wall, and that this function depends on proper glycosylation of the molecules. As a consequence, the glycoproteins are essential for growth of the pollen tube.

Regulation of flavonol biosynthesis during anther and pistil development, and during pollen tube growth in Solanum tuberosum.

The regulation of flavonol biosynthesis was studied in anthers and pistils of Solanum tuberosum. Flavonols are essential for functional pollen tube growth in a number of species. Flavonol accumulation in whole anthers started at the unicellular stage of pollen development and continued until pollen maturity. A cDNA clone encoding flavonol synthase (FLS) was isolated. Fls gene expression was detected in pistils, anthers, petals and ovaries, the organs in which flavonols are accumulating. Fls transcripts were present in unicellular and bicellular pollen, but not in mature pollen. The expression patterns of three genes encoding enzymes in the flavonoid biosynthetic pathway, chalcone synthase (chs), flavanone-3-hydroxylase and fls were analysed in developing anthers and pistils. Only chs transcripts accumulated concomitantly with the flavonols in anthers. In pistils of potato, pollen tube growth induced an increase in fls gene expression that, unlike the situation in pollinated pistils of petunia, did not result in an increased flavonol content. Flavonol biosynthesis in anthers is probably initiated by the expression of the chs gene, and flavonol accumulation in pistils upon pollen tube growth is not an universal phenomenon.

Analysis of microspore-specific promoters in transgenic tobacco.

In order to modify the early stages of pollen development in a transgenic context microspore-specific promoters are required. We tested two putatively microspore-specific promoters, the Bp4 promoter from rapeseed and the NTM19 promoter from tobacco. Expression of the gus and barnase reporter genes under the control of these two promoters was studied in transgenic tobacco. Contrary to expectations, the Bp4 promoter became active only after the first pollen mitosis, and not in the microspores. The NTM19 promoter turned out to be highly microspore-specific and directed very high levels of gus expression to the unicellular microspores. The NTM19-barnase transgene caused cell-autonomous death at the mid-unicellular microspore stage, whereas Bp4-barnase induced cell ablation of early to mid-bicellular pollen. Both promoter-barnase transgenes did not affect the sporophyte and were inherited through the female germline. These results show that both the NTM19 and Bp4 promoters are expressed only in the male germline, and that the NTM19 promoter is an excellent tool to direct high levels of transgene expression exclusively to the microspores. This may have important biotechnological applications.

Isolation and characterization of a microspore-specific gene from tobacco.

The characterization of a gene with a unique microspore-specific expression pattern is reported. Isolated microspores from tobacco were used to synthesize a cDNA library. Clones that did not hybridize to leaf cDNA were further characterized by northern analysis. One clone proved to be a microspore-specific cDNA, representing a transcript of 650 nt. The corresponding gene, NTM19 (Nicotiana tabacum microspore-specific), was isolated and its sequence analysed. The gene encodes a protein of 10.8 kDa with a pI of 6.92 and a putative signal sequence at the N-terminus. A localization study revealed a unique spatial and temporal distribution. The transcript was only detected in the unicellular microspore. No hybridization signals were observed in other pollen developmental stages, nor in the surrounding anther tissues or other vegetative tissues of the plant. Therefore it can be concluded that NTM19 is a gene with a highly microspore-specific character according to both localization and stage of expression. Southern blot analysis demonstrated the presence of a small gene family. The occurrence of TNM19 was investigated in a range of closely and distantly related species and was found to be present in other solanaceous species, including the ancestors of tobacco and in a monocot species.

Molecular analysis of a pistil-specific gene expressed in the stigma and cortex of Solanum tuberosum.

A gene, sts14, coding for a highly expressed mRNA in pistils of Solanum tuberosum, was isolated. Northern blot and in situ analyses demonstrated that the gene was expressed throughout pistil development in both the stylar cortex and the stigma. The deduced STS14 protein displays similarity to the pathogenesis-related PR-1 proteins. A possible function for protection or guidance of the pollen tubes through the pistil is discussed.

Visualization of starch-synthase expression by in situ hybridization during pollen development.

The expression of the starch-synthase gene was studied by in situ RNA hybridization at different stages of pollen development in tobacco (Nicotiana tabacum L.). By means of Northern blot analysis we demonstrated that a potato (Solarium tuberosum L.) gene was homologous to its tobacco counterpart, and then we synthesized (35)S-labeled RNA probes from this gene. The probe was seen to bind at the tetrad stage. Unicellular pollen grains showed no starch-synthase mRNA but these transcripts reappeared in the binucleate stages, being abundant in mature pollen grains. Our results show that starch-synthase is expressed in the gametophytic cells. Variations in mRNA accumulation during the different stages indicate a regulation of this gene throughout pollen development.

Stage-related expression of mRNAs during pollen development in lily and tobacco.

Homogeneous populations of developing microspores and pollen from anthers of lily (Lilium longiflorum Thumb.) and tobacco (Nicotiana tabacum L.) show a continuous production of biomass, reaching a maximum in young pollen. The rate of RNA synthesis was 460 fg · h(-1) in young binucleate cells, 138 fg · h(-1) in late binucleate cells and 56 fg · h(-1) in microspores. The mRNA population in developing pollen can be separated into three groups. In the first group, certain types of mRNAs are present at a constant level during all stages of development. A second group is characteristic of young pollen and increases quantitatively until anthesis. A third group is seen transiently; to this belong mRNAs present only before mitosis or at a distinct cell stage after mitosis. Some of the translation products of this latter group of mRNAs showed similarities between lily and tobacco on two-dimensional gels in respect of molecular weight and isolectric point, indicating that those mRNAs and proteins play a role in the regulation of pollen development.

Post-fusion incompatibility in Physarum polycephalum. The involvement of DNA.

The lethal reaction following fusion of plasmodia of a sensitive and a killer strain of Physarum polycephalum could be prevented by the incorporation of 5-bromo-2'-deoxyuridine in both strains. This result suggests the involvement of transcription in the lethal reaction. Although under appropriate conditions fusion of the strains is not followed by a lethal reaction the fused plasmodium will behave as the killer strain after subculturing. At different times after fusion, DNA was isolated from a plasmodium in which visible lethal reaction was prevented and separated on CsCl gradients. Killer DNA remained intact but sensitive DNA was broken down, mainly from 8--11 h after fusion.

Cell-free protein synthesis with polysomes from germinating Petunia pollen grains.

1. A cell-free protein synthesizing system was developed, which is able to incorporate amino acids into protein programmed by polysomes from pollen grains. 2. The enzyme system used was a 100,000 g supernatant from rat liver. 3. The characteristics of this mixed "plant-animal" system are described 4. Ungerminated pollen contains only monosomes which have no incorporation activity in the cell-free system. 5. Formation of polysomes starts immediately with the germination accompanied by decrease of the monosomal pool. 6. The rapid synthesis of polysomes suggests the presence of a preformed, masked form of messenger-RNA (mmRNA) in the resting pollen grain. 7. The instantaneous activation of protein synthesis enables pollen germination to proceed to a stage at which the pollen genome is not yet activated.