Richard Feron

Until recently, little knowledge existed about the molecular mechanisms regulating male gamete development. This was mainly due to the low transcriptional activity and the cellular inaccessibility of the generative and sperm cells that are enclosed by the vegetative cell in pollen. In order to study sperm cell development and possible preferential fusion during double fertilization, we have constructed a cDNA library of mRNA isolated from pure tobacco sperm cells. An initial screen of 396 clones from this library has yielded 2 cDNAs representing sperm-cell-expressed transcripts, designated NtS1 and NtS2. A preliminary characterization of these two clones showed that they accumulate in both the generative and sperm cells (i.e. the male gamete) indicating that gene expression programs between these two cell types overlap. In addition, we found that NtS1 codes for a polygalacturonase suggesting a role for this enzyme in wall degradation during differentiation of the male germ cells in tobacco. Together, these results show that with the construction of this sperm-cell cDNA library we now have a powerful tool to investigate male gamete development and function.

Solanum dulcamara (bittersweet) is one of the few native species of Solanum present in Europe. It is a common weed that occupies a wide range of habitats and is often found in the direct vicinity of cultivated potatoes (Solanum tuberosum), where it could transmit diseases. A broad sampling of European S. dulcamara accessions was carried out to gain insight into the population structure and crossing preferences of this species. Three amplified fragment length polymorphism (AFLP®) primer combinations generating 288 polymorphic fragments were used to analyze 79 bittersweet accessions (245 individuals). Dendrograms revealed a low level of genetic polymorphism in the bittersweet populations, caused partially by the out-crossing nature of this species.

Auxin response factors (ARFs) are encoded by a gene family of transcription factors that specifically control auxin-dependent developmental processes. A tomato ARF gene, homologous to Arabidopsis NPH4/ARF7 and therefore designated as Solanum lycopersicum ARF7 (SlARF7), was found to be expressed at a high level in unpollinated mature ovaries. More detailed analysis of tomato ovaries showed that the level of SlARF7 transcript increases during flower development, remains at a constant high level in mature flowers, and is down-regulated within 48 h after pollination. Transgenic plants with decreased SlARF7 mRNA levels formed seedless (parthenocarpic) fruits. These fruits were heart-shaped and had a rather thick pericarp due to increased cell expansion, compared with the pericarp of wild-type fruits. The expression analysis, together with the parthenocarpic fruit phenotype of the transgenic lines, suggests that, in tomato, SlARF7 acts as a negative regulator of fruit set until pollination and fertilization have taken place, and moderates the auxin response during fruit growth.

The developmental expression pattern and localization of calreticulin were studied in Nicotiana tabacum L. anthers, pollen and pollen tubes. High transcript and protein levels were detected throughout anther development. Immunolocalization of calreticulin in the anthers showed particular dense label in tapetum and pollen at developmental stage 2, when the tapetum is highly active and the pollen tetrads are formed. Much lower transcript and protein levels were detected in dry and hydrated pollen and in pollen tubes. Immunofluorescence labeling of both chemically fixed and cryo-fixed and freeze-substituted pollen tubes showed the presence of calreticulin in Golgi apparatus and endoplasmic reticulum (ER). Calreticulin was seen throughout the stacks in the Golgi apparatus and in the areas with coated-Golgi vesicles but much less so in the ER. Calreticulin was not found in the secretory vesicles. A relatively intense label was occasionally seen adjacent to the wall of the tube. No significant label was observed in mitochondria, vacuoles, generative cells, cell wall or callose plugs. The present results are consistent with a role of calreticulin in Ca2+-dependent folding of secreted glycoproteins in tapetum, pollen and pollen tubes.

Using differential screening we isolated a pistil-specific cDNA clone corresponding to a 1.2 kb mRNA and encoding a 32.5 kDa protein. The amino acid sequence shared similarity with that of group-I grass pollen allergens, which are known to have expansin activity. This clone, which later showed to share homology also with β-expansins, was named PPAL. The PPAL mRNA was specifically expressed in the secretory zone of the stigma and in the epidermal layer of the placenta. The accumulation level of the transcript increased during pollination, and the protein was secreted in the stigmatic exudate of the tobacco flower. We suggest here that PPAL is a new expansin, acting as a cell-wall-loosening agent during pollination.

Initial screening of 14 Solanum dulcamara accessions enabled the identification of individuals resistant and susceptible to Phytophthora infestans. Crosses between contrasting genotypes resulted in three F2–BC1 populations segregating for resistance to late blight in a laboratory assay and under field conditions. Genetic profiling of one of these populations using 128 AFLP primers generated three markers linked to the resistant phenotype. Blast analysis of the sequenced markers resulted in a plausible gene position on the distal end of the long arm of chromosome 9 that could be confirmed by CAPS markers. Thus, we describe a first resistant gene, named Rpi-dlc1, from S. dulcamara, a Solanum species native to Europe. In addition, one population was tested for broadness of resistance responses using a set of seven additional P. infestans isolates, varying in virulence. This indicated the possible presence of additional Rpi genes.

• Plant hormones are considered to be important mediators of the fruit developmental signal after pollination. The role of phytohormones in tomato (Solanum lycopersicum) fruit set was investigated here.• Transcriptome analysis of ovaries was performed using two complementary approaches: cDNA–amplified fragment length polymorphism (AFLP) and microarray analysis.• The gene expression profiles obtained suggest that, in addition to auxin and gibberellin, ethylene and abscisic acid (ABA) are involved in regulating fruit set. Before fruit development, many genes involved in biotic and abiotic responses are active in the ovary. In addition, genes involved in ethylene and ABA biosynthesis were strongly expressed, suggesting relatively high ethylene and ABA concentrations before fruit set. Induction of fruit development, either by pollination or by gibberellin application, attenuated expression of all ethylene and ABA biosynthesis and response genes within 24 h.• It is proposed that the function of ABA and ethylene in fruit set might be antagonistic to that of auxin and gibberellin in order to keep the ovary in a temporally protected and dormant state; either to protect the ovary tissue or to prevent fruit development before pollination and fertilization occur.Plant hormones are considered to be important mediators of the fruit developmental signal after pollination. The role of phytohormones in tomato (Solanum lycopersicum) fruit set was investigated here.Transcriptome analysis of ovaries was performed using two complementary approaches: cDNA–amplified fragment length polymorphism (AFLP) and microarray analysis.The gene expression profiles obtained suggest that, in addition to auxin and gibberellin, ethylene and abscisic acid (ABA) are involved in regulating fruit set. Before fruit development, many genes involved in biotic and abiotic responses are active in the ovary. In addition, genes involved in ethylene and ABA biosynthesis were strongly expressed, suggesting relatively high ethylene and ABA concentrations before fruit set. Induction of fruit development, either by pollination or by gibberellin application, attenuated expression of all ethylene and ABA biosynthesis and response genes within 24 h.It is proposed that the function of ABA and ethylene in fruit set might be antagonistic to that of auxin and gibberellin in order to keep the ovary in a temporally protected and dormant state; either to protect the ovary tissue or to prevent fruit development before pollination and fertilization occur.

This study was aimed at examining the relationships between the African material of Solanum americanum (also designated as S. nodiflorum), accessions of this taxon from other geographical areas, and American S. americanum using AFLP markers. 96 individuals representing 39 accessions of S. americanum sensu lato and related diploid species from the widest possible geographical range, and one accession of S. dulcamara (as outgroup) were used. The AFLP results suggested that American S. americanum differs from S. nodiflorum and that the material investigated in this study can be assigned to three different species: S. americanum sensu stricto, S. nodiflorum and a Solanum species from Brazil. These species can be differentiated based on a combination of floral and fruit characteristics.

The class III pistil-specific extensin-like proteins (PELPIII) of Nicotiana tabacum accumulate in the intercellular matrix (IM) of the style transmitting tissue (TT). After pollination, the 110–140 kDa PELPIII is translocated from the IM into the pollen tube walls. PELPIII-like sequences have been found in several solanaceous species. These sequences are expressed in mature non-pollinated styles at both RNA and protein level. Of the genus Nicotiana, the species N. alata, N. x sanderae and N. sylvestris (section Alatae), and N. tomentosiformis and N. otophora (section Tomentosae) showed an expression level of PELPIII homologues similar to that in mature styles of N. tabacum. PELPIII genes were absent in the most ancient species studied, namely N. trigonophylla (section Trigonophyllae). To study the species dependence of the translocation of PELPIII into the pollen tube wall in tobacco, interspecific pollinations on N. tabacum pistils were carried out with pollen from the incongruous species N. rustica, N. trigonophylla and Petunia hybrida, where PELPIII homologues are absent in the style. Immunocytological tests showed that the N. tabacum PELPIII is translocated into the pollen tube walls of all three species. Thus, the pollen tube walls of these species do not form a barrier for IM compounds such as the 110–140 kDa PELPIII and the absence of any possible effect of PELPIII on pollen tube growth cannot be due to failure of PELPIII transport through the wall. The importance of these findings is discussed with respect to the evolutionary origin of PELPIII, the pollen pistil interaction, the function of style TT-specific proteins and the physical properties of pollen tube walls.

Although the hormones, gibberellin and auxin, are known to play a role in the initiation of fruits, no such function has yet been demonstrated for abscisic acid (ABA). However, ABA signaling and ABA responses are high in tomato (Solanum lycopersicum L.) ovaries before pollination and decrease thereafter (Vriezen et al. in New Phytol 177:60–76, 2008). As a first step to understanding the role of ABA in ovary development and fruit set in tomato, we analyzed ABA content and the expression of genes involved in its metabolism in relation to pollination. We show that ABA levels are relatively high in mature ovaries and decrease directly after pollination, while an increase in the ABA metabolite dihydrophaseic acid was measured. An important regulator of ABA biosynthesis in tomato is 9-cis-epoxy-carotenoid dioxygenase (LeNCED1), whose mRNA level in ovaries is reduced after pollination. The increased catabolism is likely caused by strong induction of one of four newly identified putative (+)ABA 8′-hydroxylase genes. This gene was named SlCYP707A1 and is expressed specifically in ovules and placenta. Transgenic plants, overexpressing SlCYP707A1, have reduced ABA levels and exhibit ABA-deficient phenotypes suggesting that this gene encodes a functional ABA 8′-hydroxylase. Gibberellin and auxin application have different effects on the LeNCED1 and SlCYP707A1 gene expression. The crosstalk between auxins, gibberellins and ABA during fruit set is discussed.