In order to characterize regulatory genes that are expressed in ovule tissues after fertilization we have undertaken an EST sequencing project in Solanum chacoense, a self-incompatible wild potato species. Two cDNA libraries made from ovule tissues covering embryo development from zygote to late torpedo-stage were constructed and plated at high density on nylon membranes. To decrease EST redundancy and enrich for transcripts corresponding to weakly expressed genes a self-probe subtraction method was used to select the colonies harboring the genes to be sequenced. 7741 good sequences were obtained and, from these, 6374 unigenes were isolated. Thus, the self-probe subtraction resulted in a strong enrichment in singletons, a decrease in the number of clones per contigs, and concomitantly, an enrichment in the total number of unigenes obtained (82%). To gain insights into signal transduction events occurring during embryo development all the receptor-like kinases (or protein receptor kinases) were analyzed by quantitative real-time RT-PCR. Interestingly, 28 out of the 30 RLK isolated were predominantly expressed in ovary tissues or young developing fruits, and 23 were transcriptionaly induced following fertilization. Thus, the self-probe subtraction did not select for genes weakly expressed in the target tissue while being highly expressed elsewhere in the plant. Of the receptor-like kinases (RLK) genes isolated, the leucine-rich repeat (LRR) family of RLK was by far the most represented with 25 members covering 11 LRR classes.

Currently, the most attractive working model of gametophytic self-incompatibility (SI) involving SRNases postulates the presence of an inhibitor protein or complex expressed in pollen tubes that would counteract the cytotoxic effect of the ribonuclease activity of the SRNase. Since it has been previously shown that allelespecific recognition is mediated through the hypervariable domain sequence of the S-RNase, we have targeted this region to isolate pollen-expressed interacting proteins in the yeast two-hybrid system. One of the isolated proteins corresponds to a RING finger protein highly similar to the previously isolated SBP1 protein from Petunia hybrida. This protein is postulated to be part of the RING finger E3 ligase family. The ScSBP1 gene is expressed in almost all tissues tested, suggesting a more general role than only being involved in SI. Although the ScSBP1 gene is polymorphic, linkage analysis showed that it was unlinked to the S-locus. The isolation of this S-RNase-binding protein in two different species and with four different SRNase sequences as bait, strengthens its putative involvement in the SI response. Furthermore, comparison of the bait sequences used suggests that the SBP1 protein interacts with conserved sequences located between the HVa and HVb domains.

We have isolated a plant NOTCHLESS (NLE) homolog from the wild potato species Solanum chacoense Bitt., encoding a WD-repeat containing protein initially characterized as a negative regulator of the Notch receptor in animals. Although no Notch signaling pathway exists in plants, the NLE gene is conserved in animals, plants, and yeast. Overexpression of the plant ScNLE gene in Drosophila similarly affected bristle formation when compared to the overexpression of the endogenous Drosophila NLE gene, suggesting functional conservation. Expression analyses showed that the ScNLE gene was fertilization-induced and primarily expressed in ovules after fertilization, mainly in the integumentary tapetum (endothelium). Significant expression was also detected in the shoot apex. Promoter deletion analysis revealed that the ScNLE promoter had a complex modulatory architecture with both positive, negative, and tissue specific regulatory elements. Transgenic plants with reduced levels of ScNLE transcripts displayed pleitotropic phenotypes including a severe reduction in seed set, consistent with ScNLE gene expression pattern.

The sterol 14a-demethylase (CYP51) is the most widely distributed cytochrome P450 gene family being found in all biological kingdoms. It catalyzes the first step following cyclization in sterol biosynthesis, leading to the formation of precursors of steroid hormones, including brassinosteroids, in plants. Most enzymes involved in the plant sterol biosynthesis pathway have been characterized biochemically and the corresponding genes cloned. Genes coding for enzymes promoting substrate modifications before 24-methylenelophenol lead to embryonic and seed defects when mutated, while mutants downstream the 24-methylenelophenol intermediate show phenotypes characteristic of brassinosteroid mutants. By a differential display approach, we have isolated a fertilization-induced gene, encoding a sterol 14a-demethylase enzyme, named CYP51G1-Sc. Functional characterization of CYP51G1-Sc expressed in yeast (Saccharomyces cerevisiae) showed that it could demethylate obtusifoliol, as well as nontypical plant sterol biosynthetic intermediates (lanosterol), in contrast with the strong substrate specificity of the previously characterized obtusifoliol 14a-demethylases found in other plant species. CYP51G1-Sc transcripts are mostly expressed in meristems and in female reproductive tissues, where they are induced following pollination. Treatment of the plant itself with obtusifoliol induced the expression of the CYP51G1-Sc mRNA, suggesting a possible role of this transient biosynthetic intermediate as a bioactive signaling lipid molecule. Furthermore, treatments of leaves with 14 C-labeled obtusifoliol demonstrated that this sterol could be transported in distal parts of the plant away from the sprayed leaves. Arabidopsis (Arabidopsis thaliana) CYP51 homozygous knockout mutants were also lethal, suggesting important roles for this enzymatic step and its substrate in plant development.Sterols are ubiquitous components of the plasma membrane, where they play an important role in membrane fluidity and permeability (Hartmann and Benveniste, 1987;Hartmann, 1998) and as precursors of the brassinosteroids (BRs), a group of plant growth regulators known to affect a wide variety of physiological processes. BRs are involved in stem elongation, root growth inhibition, leaf bending and unrolling, pollen tube growth, photomorphogenesis, tracheary element differentiation, promotion of 1-aminocyclopropane-1-carboxylic acid production, and cell elongation mediated either through microtubule reorientation or through alteration of the mechanical properties of the cell wall (Clouse and Sasse, 1998;Clouse, 2002aClouse, , 2002b. Sitosterol, campesterol, and stigmasterol are the most abundant sterols in the plant membrane. More than 30 enzymatic steps catalyze the sequential reactions of sterol biosynthesis (Benveniste, 2004), resulting in the wide variety of sterols and biosynthetic intermediates found in plants. More than 60 sterols and derivatives have been identified in maize (Zea mays) seedlings (Guo et al., 1995), while animals and fungi contain a ...

WD-repeat proteins are involved in a breadth of cellular processes. While the WD-repeat protein encoding gene NOTCHLESS has been involved in the regulation of the Notch signaling pathway in Drosophila, its yeast homolog Rsa4p was shown to participate in 60S ribosomal subunit biogenesis. The plant homolog ScNLE was previously characterized in Solanum chacoense (ScNLE) as being involved in seed development. However, expression data and reduced size of ScNLE underexpressing plants suggested in addition a role during shoot development. We here report the detailed phenotypic characterization of ScNLE underexpressing plants during shoot development. ScNLE was shown to be expressed in actively dividing cells of the shoot apex. Consistent with this, ScNLE underexpression caused pleiotropic defects such as a reduction in aerial organ size, a reduction in some organ numbers, delayed flowering, and an increase in stomatal index. Analysis of adaxial epidermal cells revealed that both cell number and cell size were reduced in mature leaves of ScNLE underexpressing lines. Two-hybrid screens with the Nle domain and the WD-repeat domain of ScNLE allowed the isolation of homologs of yeast MIDASIN and NSA2 genes, the products of which are involved in 60S ribosomal subunit biogenesis in yeast. A ScNLE-GFP chimeric protein was localized in both the cytoplasm and nucleus. These data altogether suggest that ScNLE likely plays a role in 60S ribosomal subunit biogenesis, which is essential for proper cellular growth and proliferation during plant development.

Female gametophyte development in Arabidopsis thaliana follows a well-defined program that involves many fundamental cellular processes. In this study, we report the involvement of the Arabidopsis thaliana MIDASIN1 (AtMDN1) gene during female gametogenesis through the phenotypic characterization of plants heterozygous for an insertional mdn1 mutant allele. The MDN1 yeast ortholog has previously been shown to encode a non-ribosomal protein involved in the maturation and assembly of the 60S ribosomal subunit. Heterozygous MDN1/mdn1 plants were semisterile and mdn1 allele transmission through the female gametophyte was severely affected. Development of mdn1 female gametophyte was considerably delayed compared to their wild-type siblings. However, delayed mdn1 female gametophytes were able to reach maturity and a delayed pollination experiment showed that a small proportion of the female gametophytes were functional. We also report that the Arabidopsis NOTCHLESS (AtNLE) gene is also required for female gametogenesis. The NLE protein has been previously shown to interact with MDN1 and to be also involved in 60S subunit biogenesis. The introduction of an AtNLE-RNA interference construct in Arabidopsis led to semisterility defects. Defective female gametophytes were mostly arrested at the one-nucleate (FG1) developmental stage. These data suggest that the activity of both AtMDN1 and AtNLE is essential for female gametogenesis progression.

Fertilization triggers a unique and complex developmental program leading to embryogenesis and seed set. Recently, mutations affecting chromatin-remodeling enzymes in plants have shown their key roles in development as demonstrated before in animal cells. Using a negative selection screen to isolate genes expressed in ovary tissues upon fertilization, we have identified a histone deacetylase gene (named ScHD2a ) of the plant-specific HD2 family, which is predominantly expressed in ovaries of the self-incompatible species Solanum chacoense. The ScHD2a is the probable orthologue of the Arabidopsis thaliana AtHD2a gene, which upon antisense suppression leads to aborted seeds formation. Transcription of the ScHD2a gene is strongly triggered by fertilization and transcripts accumulate predominantly in the micropylar region of the ovule's integument. Interestingly, this fertilization-induced accumulation pattern was also observed for other genes involved in transcriptional repression but not for a MYST-family histone acetyltransferase. The strong increase in ScHD2a mRNA levels in ovules after fertilization suggests an important and localized role for transcriptional repression in seed development, and indicates why silencing of the AtHD2a gene leads to aborted seed formation.

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