No abstract

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.

Regulatory elements within the promoter of the pollen-specific NTP303 gene from tobacco were analysed by transient and stable expression analyses. Analysis of precisely targeted mutations showed that the NTP303 promoter is not regulated by any of the previously described pollen-specific cis-regulatory elements. However, two adjacent regions from -103 to -86 bp and from -86 to -59 bp were shown to contain sequences which positively regulated the NTP303 promoter. Both of these regions were capable of driving pollen-specific expression from a heterologous promoter, independent of orientation and in an additive manner. The boundaries of the minimal, functional NTP303 promoter were determined to lie within the region -86 to -51 bp. The sequence AAATGA localized from -94 to -89 bp was identified as a novel cis-acting element, of which the TGA triplet was shown to comprise an active part. This element was shown to be completely conserved in the similarly regulated promoter of the Bp 10 gene from Brassica napus encoding a homologue of the NTP303 gene.

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.

The pollen-specific gene Ntp303 belongs to the class of late pollen specific genes. It is first transcribed directly after pollen mitosis. Biochemical properties, appearance and precise location of the NTP303 protein during pollen development and pollen tube growth were studied by amino-acid micro-sequencing, protein gel blotting and immuno-localization. Antisera were raised against recombinant proteins, encoded by sequences of the pollen-specific Ntp303 gene. The antibodies specifically recognized a 69-kDa glycoprotein. Electron-microscopic immuno-localization of the protein revealed the presence of high concentrations of the NTP303 protein at the vegetative plasma membranes that surround the vegetative cell, the generative cell and the sperm cells of pollen and pollen tubes. The generative plasma membranes of the generative cell and the sperm cells were negative. NTP303 protein was also present in the cell walls and in callose plugs. With this method it was shown that the NTP303 protein was already present in mid-bicellular pollen, after the first, asymmetrical pollen mitosis. Possible functions for the NTP303 protein are discussed in relation to its properties and its association with the vegetative plasma membranes.

Using a Clark micro electrode the actual oxygen tension at various places in a style can be measured. Within the unpollinated, ripe style of Hippeastrum a pO2 gradient occurs. In the upper part, possibly with a little increase in the stigmatic branches, a very high oxygen tension is found; in the lower part, about 5 mm from the ovary, a sudden decrease occurs. In the ovary itself the oxygen tension is extremely low. After pollination, passage of the mass of pollen tube tips can be followed by a fairly sharp drop of the oxygen tension, which shifts down the style with progressive growth. This oxygen tension depression seems to be sharply limited to the region of pollen tube tips and the amount of decrease is dependent on the growth rate of the tubes. After passage of the pollen the original high level of oxygen tension is not fully restored in the upper style. This suggests that the metabolic activity of the pollen tube is strongly linked with the metabolic condition in the style. It seems that tubes grow most of their way to the egg under aerobic conditions; only at the base of the style, just before entering into the ovary, does the pollen have to switch over to an anaerobic pathway. For most of the style oxygen tension does not form a tropic gradient for tube growth.

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

Synchronously germinating Petunia pollen in an aerated sucrose medium release free amino acids during the first minutes after contact with the aqueous medium. The release process between 1 minute and 6 hours follows an exponential equation. The content of free amino acids of the germinating pollen decreases sharply. Free amino acids set free from "dead" pollen reach their plateau after 40 minutes in the alcoholic extraction medium. Differences in the amino acid curves show that only a part of them is localized in the pollen wall, but the majority are peptide bound in the inner space of the microspore. Consideration of the data obtained by differential analysis of the amino acid curves suggests that amino acids and protein are synthesized in the early phase of pollen germination.