This report describes the isolation and characterization of a cDNA clone representing a gene specifically expressed in pollen. A cDNA library was constructed against mRNA from mature pollen of Nicotiana tabacum. It was screened differentially against cDNA from mRNA of leaf and of pollen. One clone, NTPc303, was further characterized. On northern blot this clone hybridizes to a transcript 2100 nucleotides in length. NTPc303 is abundant in pollen. Expression of the corresponding gene is restricted to pollen, because no other generative or vegetative tissue contains transcripts hybridizing to NTPc303. Expression of NTP303 is evolutionarily conserved: homologous transcripts are present in pollen from various plant species. The first NTP303 transcripts are detectable on northern blot at the early bi-nucleate stage and accumulate until the pollen has reached maturity. During germination and pollen tube growth in vitro new NTP303 transcripts appear. This transcription has been proved by northern blots as well as by pulse labelling experiments. Nucleotide sequence analysis revealed that NTPc303 has an open reading frame coding for a predicted protein of 62 kDa. This protein shares homology to ascorbate oxidase and other members of the blue copper oxidase family. A possible function for this clone during pollen germination is discussed.

SummaryThe regulation of flavonol biosynthesis was studied in anthers and pistils of Solanum tuberosum.

The effect of elevated temperatures on protein synthesis in pollen from Petunia hybrida and Lilium longiflorum was investigated and compared with their effect in root-tissue tissue of Petunia. The results demonstrate that the incorporation of [35-S] methionine into protein does not change in pollen incubated at temperatures up to 40°C, but decreased with 65% at 45°C as compared to incorporation at 27"e.The qualitative changes that occurred for the proteins synthesized at 2r and 40°C were not identical for pollen from Petunia and Lilium. The proteins synthesized at 27°and 40°C were very similarwithin one species. None of the changes in proteins synthesized at 40°C in pollen showed anycorrelation with heat shock proteins formed in rootlets from Petunia.

The localization of transcripts of the pollenspecific gene NTP303 has been determined by means of in situ hybridization in combination with confocal laser 1987; Blackm ore and Knox. 1990; Bedinger 1992; O tta viano et a l 1992), including m olecular biology (M ascarenhas 1990; M cC orm ick et al. 1991; Scott et al. scanning microscopy. NTP303 transcripts were first de-1991b; W eterings et al. 1992). The study o f pollen developm ent is of considerable agronom ic im portance for plant breeding, propagation, and seed production. A t the sam e time, it is also an intriguing as well as a convenient m odel system to study the m echanism of gene regulation. The pollen genom e is haploid and is actively transcribed. Specific transcripts in the m icrospore and pollen grain appear and disap pear during its synchronous developm ent (Schrauwen e ta l. 1990; Scott et al. 1991a), In the m ature pollen grain, the vegetative cell and the generative cell or its derivatives, the sperm cells, have their own specialized functions. Clearly the delegation of tasks to the different cells of the m ale gam etophyte must im plicate a cellspecific regulation of genes. T hese properties, together with the relative ease of isolation, m ake the pollen grain a very suitable object for m olecular biological studies. We have set out to study gene regulation during p o l len developm ent by isolating a pollen-specific c D N A clone-NTPc3Q3-from Nicotiana tabacum (Weterings In flowering plants, the male gametophyte has been re-et al. 1992). N T P 303 R N A is n ot detectable in sporo-tectable at the mid-bi-nucleate stage of pollen develop ment and persisted even after the pollen tube had reached the ovary. The majority of the NTP303 tran scripts were localized in the vegetative cell, and were predominantly present in the apex of the pollen tube. To study the mechanism of regulation, the genomic clone NTPg303 was isolated and characterized. NTPg303 be longs to a gene family of at least five members. N o in trons are present in its coding region. Regions matching the pollen-specific PB-element TG TG G TT (Twell et al. 1991) have been found.

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Summary. The application of confocal laser scanning microscopy together with in situ hybridization experiments in tobacco pollen enabled a detailed localization of a pollen-specific mRNA. The three-dimensional distribution of this specific mRNA over the whole pollen grain was reconstructed by means of optical sections of one specimen.

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