Z1943 is a EST clone selected in the preliminary screening as one candidate from the stage 3 genes and whose corresponding unique gene was named (Figure 1A)

Z1943 is a EST clone selected in the preliminary screening as one candidate from the stage 3 genes and whose corresponding unique gene was named (Figure 1A). Nevertheless, a large proportion (25% in the genome) of genes present in herb genomes still have no attributed function (Berardini et al., 2004). The goal is now to actively find the functions of such unknown genes for a better understanding of the overall processes. To define the gene functions required for a distinct developmental process, many studies have been conducted around the differentiation of common cell types. An example is the in vitro differentiation system in which freshly isolated mesophyll cells can be specifically induced to transdifferentiate into tracheary elements (TEs) by the application of auxin and cytokinin (Fukuda and Komamine, 1980). TEs form xylem vessel strands through the tissues of vascular plants to transport water, nutrients, and signaling molecules (Fukuda, 1996). To be fully functional, mature vessel elements are first strengthened with thick cell walls, called secondary cell walls (SCWs), which are laid down beneath thin primary cell walls in some Rabbit polyclonal to Smac specialized cells and are composed mostly of cellulose, hemicellulose, and lignin; they then drop their cellular content and are finally perforated, thereby forming a reinforced hollow cylinder with an accessible lumen suitable for xylem sap conduction (Aloni, 1987; Turner et al., 2007). To unravel the specific gene expression associated with TE formation, EST sequencing and microarray expression profiling have Vc-seco-DUBA been performed (Demura et al., 2002; Milioni et al., 2002; Pesquet et al., 2005). Although the TE differentiation system represents an excellent source of gene candidates, functional analysis of these genes during the differentiation process remained inaccessible until recently because an effective method of direct transformation of the in vitro system has only just been developed (Endo et al., 2008). Using this method, nucleic acids, including plasmid DNAs and double-stranded RNAs (dsRNAs), can be introduced and subsequently either activate gene expression or specifically silence the expression of target genes, respectively, in differentiating cells (Endo et al., 2008). Combining this recently developed transient transformation method and the global transcriptome analyses of in vitro TE differentiation, we performed an RNA interference (RNAi) screen with selected genes of unknown function expressed at specific stages of TE differentiation to identify gene candidates that directly affect TE differentiation. The candidates were further analyzed using both the in vitro system and whole plants to define the role of these unknown genes in TE differentiation. In this study, we focused on two closely related unknown genes that are expressed when TEs are forming their SCWs: (cell cultures and transgenic seedlings, we unraveled their function in SCW deposition and their potential conversation with the SCW synthesis machinery. RESULTS Screening Genes with Unknown Function by RNAi in the TE Differentiation System Using a recently developed dsRNA-mediated RNAi method (Endo et al., 2008), a preliminary genetic screen to identify gene candidates directly affecting the rate of TE differentiation was performed using genes with specifically upregulated expression at different time points along the TE differentiation time course. These genes were identified by microarray analysis and can be classified into stage 1, 2, and 3 genes based on their expression patterns during TE differentiation (Figures 1A and 1B; Demura et al., 2002). Z1943 is usually a EST clone selected in the preliminary screening as one candidate from the stage 3 genes and whose corresponding unique gene was named (Physique 1A). dsRNA-mediated RNAi for resulted Vc-seco-DUBA in a significant decrease in the number of cells with visible SCWs, which represented TEs, in the in vitro TE differentiation culture, as compared with a control using dsRNA corresponding to a partial sequence of DNA (Physique 1C). Since encodes a previously uncharacterized protein with no attributed function, we decided to analyze the function of further. Another EST clone (“type”:”entrez-nucleotide”,”attrs”:”text”:”Z16653″,”term_id”:”23216″,”term_text”:”Z16653″Z16653) was identified with significant sequence similarity to (Physique 1D; see Supplemental Physique 1A online), and its corresponding gene was thereafter named also has stage 3Cspecific expression (Physique 1B), and dsRNA-mediated RNAi for also significantly reduced the rate of SCW formation (Physique 1C). Although two alleles of and (cv Canary Bird) (see Supplemental Physique 1A online), genotyping revealed that most individual Vc-seco-DUBA plants have Vc-seco-DUBA (see Supplemental Physique 1B online). The predominant sequence was therefore used as the gene in all of the following experiments. Open in a separate.