Silencing of the pollen-specific gene NTP303 and its family members in tobacco affects in vivo pollen tube growth and results in male sterile plants.

In seed plants, successful fertilization requires correct regulation of pollen tube growth. At germination and during growth, the pollen tube interacts with tissues from the pistil while the pollen tube extends via tip growth. Despite the fact that much research has been devoted to the mechanisms regulating pollen tube growth, many aspects are currently unknown.

Regulation of anthraquinone biosynthesis in cell cultures of Morinda citrifolia.

Cell cultures of Morinda citrifolia L. are capable of accumulating substantial amounts of anthraquinones. Chorismate formed by the shikimate pathway is an important precursor of these secondary metabolites.

The 5'-untranslated region of the ntp303 gene strongly enhances translation during pollen tube growth, but not during pollen maturation.

Transcripts of the ntp303 gene accumulate abundantly throughout pollen development, whereas the protein only accumulates to detectable levels after pollen germination. In an attempt to explain the divergence in the accumulation profiles of the mRNA and the protein, we investigated the role of the untranslated regions (UTRs) in enhancing ntp303 translation during the transition from developing to germinating pollen. Luciferase reporter gene fusion constructs containing the ntp303 5'-UTR gave rise to luciferase activity that was up to 60-fold higher during pollen tube growth than that of constructs containing different 5'-UTRs.

Regulation of expression of two novel flower-specific genes from tomato (Solanum lycopersicum) by gibberellin.

Two flower-specific cDNAs have been isolated after differential screening of an anther cDNA library. This library was constructed 48 h after GA(3) treatment of buds of the GA-deficient gib-1 mutant of tomato. Northern blot analysis during flower development in tomato demonstrated that the expression of both genes is regulated by gibberellins (GAs).

The expression of tgas118, encoding a defensin in Lycopersicon esculentum, is regulated by gibberellin.

A flower specific cDNA, tgas118, has been isolated after differential screening of a gib-1 anther cDNA library of Lycopersicon esculentum. The corresponding mRNA was present in all tissues analysed. Northern blot analysis revealed that in wild-type tomato the gene was predominantly expressed throughout flower development, while in the gibberellin (GA)-deficient mutant of tomato (gib-1) the abundance declined.

Partial silencing of the NEC1 gene results in early opening of anthers in Petunia hybrida.

The NEC1 gene, previously isolated from Petunia hybrida, is expressed at high levels in nectaries, and in a very localized fashion in stamens, particularly in the anther stomium cells and the upper part of the filament. To elucidate the function of the NEC1 gene, co-suppression was employed for down-regulation of NEC1 expression, and transposon insertion mutagenesis was used to knock out the NEC1 function. Among the transgenic plants and plants carrying dTph1 inserted in the NEC1 gene, an "early open anther" phenotype was observed.

NEC1, a novel gene, highly expressed in nectary tissue of Petunia hybrida.

To study the molecular regulation of nectary development, we cloned NEC1, a gene predominantly expressed in the nectaries of Petunia hybrida, by using the differential display RT-PCR technique. The secondary structure of the putative NEC1 protein is reminiscent of a transmembrane protein, indicating that the protein is incorporated into the cell membrane or the cytoplast membrane. Immunolocalization revealed that NEC1 protein is present in the nectaries.

Isolation and molecular characterization of gibberellin-regulated H1 and H2B histone cDNAs in the leaf of the gibberellin-deficient tomato.

After differential screening we isolated cDNA clones encoding a histone H1 (leH1) and three variants of histone H2B (leH2B-1, -2 and -3) from the gibberellin (GA)-deficient mutant of tomato (gib-1). The deduced polypeptide of leH1 is 271 amino acids long and exhibits the typical tripartite structure of histones H1. The full-length cDNA clone leH2B-1 encodes for a protein of 142 amino residues and shows the tripartite organization of histones H2B.

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.

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.

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.

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.

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.

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.

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.

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.

Functional dissection of the promoter of the pollen-specific gene NTP303 reveals a novel pollen-specific, and conserved cis-regulatory element.

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.

Isolation and Characterization of Mutants of Thiophene Synthesis in Tagetes erecta.

Two mutants of Tagetes erecta displaying aberrant thiophene composition were identified by screening more than 300 plants from a mutagenized M2 population using high-performance liquid chromatography analysis of root extracts. Both mutants, which may have originated from the same mutational event, contained high amounts of the C13 monothiophene 2-(but-3-en-1-ynyl)-5-(penta-1,3-diynyl)-thiophene that was previously not found in T. erecta and also high amounts of two C13 bithienyls that were absent or present at low concentrations in the wild type.

Thiophene interconversion in elicitor-treated roots ofTagetes patula L.

Thiophenes are polyacetylene-related heterocyclic metabolites. Some of these compounds are phototoxic, but the bithiophenes occurring inTagetes mainly accumulate in the root where photo-activation is not likely to occur. A cell-free extract from the fungusFusarium oxysporum induced biosynthesis of hydrophilic thiophenes in root cultures and roots of seedlings ofTagetes patula.

Isolation and characterization of mRNAs accumulated during in-vitro flower bud formation.

The development of vegetative and generative buds on thin-layer explants of tobacco (Nicotiana tabacum L. cv. Samsun) has been studied at the level of translatable mRNA to detect changes in the mRNA population during bud initiation and differentiation, and several quantitative differences were found.

Characterization of a pollen-specific cDNA clone from Nicotiana tabacum expressed during microgametogenesis and germination.

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.

In vitro flower bud formation in tobacco: interaction of hormones.

External application of auxin and cytokinin is required for the formation of flower buds on thin-layer tissue explants of Nicotiana tabacum cv Samsun. Interaction between both plant growth regulators during this regenerative process has been demonstrated with respect to speed of flower bud initiation and the number of flower buds formed. Separation in time of the hormone application during culture revealed that the cytokinin benzyladenine plays a key role in flower bud initiation whereas auxin (indoleacetic acid) stimulates in particular the differentiation of flower buds.

Inhibition by Ethylene of Auxin-Promotion of Flower Bud Formation in Tobacco Explants Is Absent in Plants Transformed by Agrobacterium rhizogenes.

The in vitro regeneration of flower buds was studied in pedicel explants from tobacco (Nicotiana tabacum L., cv Petit Havana) transformed with Agrobacterium rhizogenes, pRi 1855 (agropine type). At a low concentration (0.

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.