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.

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.

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.

Evidence for sense RNA-mediated protection to PVYN in tobacco plants transformed with the viral coat protein cistron.

The coat protein (CP) cistron of the tobacco veinal necrosis strain of potato virus Y (PVYN), supplemented with translational start signals, was cloned into an Agrobacterium tumefaciens Ti transformation vector. Transformation of tobacco leaf discs resulted in 99 transgenic lines which were subsequently analysed for the presence and expression, at both the transcriptional and translational level, of the CP-gene. Although CP-specific RNA transcripts were produced in all plants no CP could be detected by several sensitive immunological techniques. Upon mechanical inoculation of progeny lines of self-pollinated original transformants (S1) with PVYN, protection levels of 20 and 95%, respectively, could be observed in two out of ten lines tested. This level of protection increased to 100% in the S2 progeny obtained from self-pollination of virus-protected S1 plants. Transformation of tobacco leaf discs with a PVYN CP construct from which the ATG start codon had been removed by site-directed mutagenesis resulted in 57 transgenic lines that all produced CP-specific transcripts. Mechanical inoculation with PVYN of S1 progeny plants of several of these lines resulted in resistance to a similar level and extent as in the S1 progeny of plants transformed with the intact CP cistron. The results obtained strongly suggest that the resistance observed in the transgenic plants is principally based on the presence of PVYN CP RNA sequences rather than on the accumulation of viral coat protein.