Transcription profiling of fertilization and early seed development events in a solanaceous species using a 7.7 K cDNA microarray from Solanum chacoense ovules

<p>Abstract</p> <p>Background</p> <p>To provide a broad analysis of gene expression changes in developing embryos from a solanaceous species, we produced amplicon-derived microarrays with 7741 ESTs isolated from <it>Solanum chacoense </it>ovules bearing embryos from all developmental stages. Our aims were to: 1) identify genes expressed in a tissue-specific and temporal-specific manner; 2) define clusters of genes showing similar patterns of spatial and temporal expression; and 3) identify stage-specific or transition-specific candidate genes for further functional genomic analyses.</p> <p>Results</p> <p>We analyzed gene expression during <it>S. chacoense </it>embryogenesis in a series of experiments with probes derived from ovules isolated before and after fertilization (from 0 to 22 days after pollination), and from leaves, anthers, and styles. From the 6374 unigenes present in our array, 1024 genes were differentially expressed (≥ ± 2 fold change, p value ≤ 0.01) in fertilized ovules compared to unfertilized ovules and only limited expression overlap was observed between these genes and the genes expressed in the other tissues tested, with the vast majority of the fertilization-regulated genes specifically or predominantly expressed in ovules (955 genes). During embryogenesis three major expression profiles corresponding to early, middle and late stages of embryo development were identified. From the early and middle stages, a large number of genes corresponding to cell cycle, DNA processing, signal transduction, and transcriptional regulation were found. Defense and stress response-related genes were found in all stages of embryo development. Protein biosynthesis genes, genes coding for ribosomal proteins and other components of the translation machinery were highly expressed in embryos during the early stage. Genes for protein degradation were overrepresented later in the middle and late stages of embryo development. As expected, storage protein transcripts accumulated predominantly in the late stage of embryo development.</p> <p>Conclusion</p> <p>Our analysis provides the first study in a solanaceous species of the transcriptional program that takes place during the early phases of plant reproductive development, including all embryogenesis steps during a comprehensive time-course. Our comparative expression profiling strategy between fertilized and unfertilized ovules identified a subset of genes specifically or predominantly expressed in ovules while a closer analysis between each consecutive time point allowed the identification of a subset of stage-specific and transition-specific genes.</p

Characterization of ScORK28, a transmembrane functional protein receptor kinase predominantly expressed in ovaries from the wild potato species Solanum chacoense

AbstractSolanum chacoense ovule receptor kinase 28 (ScORK28) was found among 30 receptor kinases from an ovule cDNA library enriched for weakly expressed mRNAs. This LRR-RLK displayed high level of tissue specificity at the RNA and protein levels and was predominantly expressed in female reproductive tissues. Protein expression analyses in planta revealed that ScORK28 was N-glycosylated and ScORK28::GFP fusion analyses showed that it was localized at the plasma membrane. Bacterial expression of ScORK28 catalytic domain followed by kinase activity assays revealed that ScORK28 is an active Mg2+-dependent protein kinase and that the juxtamembrane domain is necessary for kinase activity

Development of an efficient cis-trans-cis ribozyme cassette to inactivate plant genes

Summary Inactivation of a targeted gene is one of the main strategies used to understand their precise cellular role. In plants, apart from chemical or physical mutagenesis and random insertions of DNA elements followed by screening for a desired phenotype, the most common strategy to inhibit the expression of a given gene involves RNA silencing. This can be achieved either through antisense suppression, sense over-expression leading to co-suppression, or expression of double-stranded DNA constructs (dsRNA). The use of ribozymes to inhibit gene product accumulation has only been occasionally attempted, mainly because of the more complex genetic engineering procedure involved, although the specificity of ribozymes can be an important factor when targeting close members of a gene family. We report here the development of a new cis -acting ribozyme cassette for the production of RNAs with desired termini. Attention to many details has been brought in order to provide a powerful procedure for plant application. For example, ultrastable GNRA tetraloops were substituted for both loops II and III of cis -acting hammerhead sequences, thereby favouring folding into the catalytically active structure that results in the self-cleavage of all transcripts. We demonstrate the usefulness of this cassette by producing a ribozyme that cleaves in trans , originally embedded in the cis -acting self-cleaving cassette. The activity of the cistrans-cis construct, was demonstrated both in vitro and in vivo , in transgenic plants with the specific cleavage of an mRNA encoding a 2-oxo-glutarate-dependant dioxygenase predominantly expressed in pistils tissues and in leaves, from the wild potato Solanum chacoense

Two highly divergent alcohol dehydrogenases of melon exhibit fruit ripening-specific expression and distinct biochemical characteristics

Alcohol dehydrogenases (ADH) participate in the biosynthetic pathway of aroma volatiles in fruit by interconverting aldehydes to alcohols and providing substrates for the formation of esters. Two highly divergent ADH genes (15% identity at the amino acid level) of Cantaloupe Charentais melon (Cucumis melo var. Cantalupensis) have been isolated. Cm-ADH1 belongs to the medium-chain zinc-binding type of ADHs and is highly similar to all ADH genes expressed in fruit isolated so far. Cm-ADH2 belongs to the short-chain type of ADHs. The two encoded proteins are enzymatically active upon expression in yeast. Cm-ADH1 has strong preference for NAPDH as a co-factor, whereas Cm-ADH2 preferentially uses NADH. Both Cm-ADH proteins are much more active as reductases with Kms 10–20 times lower for the conversion of aldehydes to alcohols than for the dehydrogenation of alcohols to aldehydes. They both show strong preference for aliphatic aldehydes but Cm-ADH1 is capable of reducing branched aldehydes such as 3-methylbutyraldehyde, whereas Cm-ADH2 cannot. Both Cm-ADH genes are expressed specifically in fruit and up-regulated during ripening. Gene expression as well as total ADH activity are strongly inhibited in antisense ACC oxidase melons and in melon fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. These data suggest that each of the Cm-ADH protein plays a specific role in the regulation of aroma biosynthesis in melon fruit

Expression and trans-specific polymorphism of self-incompatibility RNases in Coffea (Rubiaceae)

Self-incompatibility (SI) is widespread in the angiosperms, but identifying the biochemical components of SI mechanisms has proven to be difficult in most lineages. Coffea (coffee; Rubiaceae) is a genus of old-world tropical understory trees in which the vast majority of diploid species utilize a mechanism of gametophytic self-incompatibility (GSI). The S-RNase GSI system was one of the first SI mechanisms to be biochemically characterized, and likely represents the ancestral Eudicot condition as evidenced by its functional characterization in both asterid (Solanaceae, Plantaginaceae) and rosid (Rosaceae) lineages. The S-RNase GSI mechanism employs the activity of class III RNase T2 proteins to terminate the growth of "self" pollen tubes. Here, we investigate the mechanism of Coffea GSI and specifically examine the potential for homology to S-RNase GSI by sequencing class III RNase T2 genes in populations of 14 African and Madagascan Coffea species and the closely related self-compatible species Psilanthus ebracteolatus. Phylogenetic analyses of these sequences aligned to a diverse sample of plant RNase T2 genes show that the Coffea genome contains at least three class III RNase T2 genes. Patterns of tissue-specific gene expression identify one of these RNase T2 genes as the putative Coffea S-RNase gene. We show that populations of SI Coffea are remarkably polymorphic for putative S-RNase alleles, and exhibit a persistent pattern of trans-specific polymorphism characteristic of all S-RNase genes previously isolated from GSI Eudicot lineages. We thus conclude that Coffea GSI is most likely homologous to the classic Eudicot S-RNase system, which was retained since the divergence of the Rubiaceae lineage from an ancient SI Eudicot ancestor, nearly 90 million years ago.United States National Science Foundation [0849186]; Society of Systematic Biologists; American Society of Plant Taxonomists; Duke University Graduate Schoolinfo:eu-repo/semantics/publishedVersio

Lipid Signaling in Plants. Cloning and Expression Analysis of the Obtusifoliol 14α-Demethylase from Solanum chacoense Bitt., a Pollination- and Fertilization-Induced Gene with Both Obtusifoliol and Lanosterol Demethylase Activity

The sterol 14α-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 14α-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 14α-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