M.M.A. van Herpen

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.

Wide single-mode tuning of a 3.0-3.8-mu m, 700-mW, continuous-wave Nd : YAG-pumped optical parametric oscillator based on periodically poled lithium niobate

A new optical parametric oscillator (OPO) for the mid-infrared wavelength region of 3-3.8mum with an idler output power of up to 1.5 W has been developed. The singly resonant OPO is pumped by a single-mode, 10-W, continuous-wave Nd:YAG laser and consists of a bow-tie ring cavity with a fan-out periodically poled lithium niobate crystal and a low-finesse intracavity air-spaced etalon. The single-frequency idler output can be continuously tuned over 24 GHz with 700-mW power by tuning of the pump laser. The tuning was demonstrated by recording of an absorption line of ethane with photoacoustic spectroscopy.

Molecular analysis of a pistil-specific gene expressed in the stigma and stylar 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.

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.

A pistil-specific gene of Solanum tuberosum is predominantly expressed in the stylar cortex

In a program aimed at studying genes expressed in pistils, the cDNA clone STS15 was isolated from a cDNA library of pollinated pistils of Solanum tuberosum and was found to be expressed only in pistils. During development of the pistil, the accumulation of STS15 transcripts, which are 0.7 kb long, reached a maximum just before anthesis and declined in fully open flowers. Southern blot analysis revealed that sts15 was present as a small gene family in dihaploid potato. In situ hybridization experiments indicated that STS15 was strongly expressed in the cortex of the style and at a low level in the stigma. No hybridization signal was observed in the transmitting tissue. The temporal and spatial expression patterns of STS15 indicate that the gene products of the sts15 gene might be involved in the function of the stylar cortex or in making the pistil competent for pollination.

Heat Shock Proteins and Survival of Germinating Pollen of Lilium longiflorum and Nicotiana tabacum

Summary Protein synthesis as well as pollen tube development of pollen of Lilium longiflorum and Nicotiana tabacum , incubated in vitro , are affected by a heat shock applied after germination for 30 min. Pollen tube elongation ceases and a protrusion is formed at the tip. The protein pattern in germinating pollen of lily is slightly changed by heat shock; cordycepin inhibited heat shock-induced de novo synthesis of two proteins, indicating that heat shock-induced regulation occurs at the level of transcription. In tobacco, however, the change in the pattern of protein synthesis is more pronounced, and the production of more heat shock proteins (HSPs) could be established. Some of those HSPs in tobacco are already present before heat shock: their synthesis at the control temperature of 27 °C can be inhibited by cordycepin, indicating that transcriptionally controlled synthesis of HSP-mRNAs occurs during normal pollen tube growth. Furthermore, some of the heat shock induced changes in the overall pattern of protein synthesis in tobacco seem to be similar to those in lily. The alterations in protein synthesis due to heat shock gradually disappear during the subsequent period of 1.5 h following heat shock. This period is characterized both by the recovery of the synthesis of the normal proteins and the restart of pollen tube growth. The recovery of the overall pattern of protein synthesis can be retarted by cordycepin as is the restart of the pollen tube growth. In lily and tobacco, a pre-heat shock of 3 min immediately after imbibition has a similar effect on the protein pattern as cordycepin: no synthesis of the HSPs when the actual heat shock of 30 min is applied.

Molecular characterization of the pollen-specific genomic clone NTPg303 and in situ localization of expression

The localization of transcripts of the pollenspecific gene NTP303 has been determined by means of in situ hybridization in combination with confocal laser scanning microscopy. NTP303 transcripts were first detectable at the mid-bi-nucleate stage of pollen development and persisted even after the pollen tube had reached the ovary. The majority of the NTP303 transcripts 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 belongs to a gene family of at least five members. No introns are present in its coding region. Regions matching the pollen-specific PB-element TGTGGTT (Twell et al. 1991) have been found.

Induced ADH gene expression and enzyme activity in pollinated pistils of Solanum tuberosum

Abstract The regulation of alcohol dehydrogenase (ADH) in relation to in vivo pollen tube growth of Solanum tuberosum was investigated. Adh gene expression as well as ADH enzyme activity were induced in pollinated pistils. The induced ADH isozyme in pollinated pistils is not present in pollen or anthers. The same ADH isozyme is induced in leaves submerged in water. The significance of the induction of ADH activity for pollen tube growth is discussed.

Cellular localization of a pollen-specific mRNA by in situ hybridization and confocal laser scanning microscopy

SummaryThe 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.