The flowering plant Arabidopsis thaliana has become an important model system with a number of desirable features such as a small genome, short generation time, small size, high seed yield, and ease of transformation (Meinke et al., 1998). Sequencing of the complete Arabidopsis thaliana genome revealed that only 9% of the 25,500 predicted genes have been attributed functions based on experimental evidence (The Arabidopsis Genome Initiative, 2000). Techniques that attribute gene function are diverse and include sequence similarity, expression profiling, proteomics, and loss- or gain-of-function mutants. It is predicted that by combining these techniques, the function of every Arabidopsis gene will be known by 2010 (Chory et al., 2000). As a part of this common goal, we have produced a catalogue of loss- and gain-of-function transgenics, which we hope will
attribute functions to uncharacterised Arabidopsis genes.
The most common approaches used to produce loss- and gain-of-function mutants rely on random insertion of DMA elements that interrupt (loss-of-function) or augment (gain-of-function) gene expression. Selection of Arabidopsis as a model system spurred the development of new techniques and publicly available resources. We have specifically taken advantage of the annotated cDNA libraries produced by EST projects (Newman et al., 1994). These resources facilitate transcript-specific loss- and gain-of-function approaches by antisense and sense, techniques usually confined to the study Of individual or a few genes. We decided to target the protein kinase family as it contains almost 1,000 (mostly uncharacterised) members, is a key component of signal transduction, and has been implicated in a wide range of biological processes. We expected antisense to
be effective in overcoming two of the problems associated with finding protein kinase function: 1. Many protein kinase genes are highly homologous (and possibly redundant); and 2. Some protein kinases have vital roles for cell viability (Meissner et al., 1999). Antisense constructs have been shown to simultaneously down-regulate the target and highly homologous genes (Mol et al., 1990), while the range of down-regulation efficiency may avoid lethal phenotypes (Bourque, 1995). Additionally, we selected some Arabidopsis genes of unknown function, with the aim of attributing biological roles for genes from this anonymous category.
To reduce the labour associated with transcript specific constructs, we produced custom-made Basta binary vectors containing the CaMV35S promoter and nopaline synthase terminator (Mylne and Botella, 1998). They allowed the direct sub-cloning of EST clones from the PRL-2 cDNA
library (Newman et al., 1994) in the antisense or sense orientation. Using these constructs, simple transformation protocols (Bechtold, Ellis, and Pelletier, 1993) and Basta selection of transformants on soil, 67 EST clones (53 belonging to the protein kinase family and 14 of unknown function) were used to produce 1,443 independent antisense transgenic lines. 22 EST clones (13 unknowns, 6 protein kinases and 3 phosphatases) that were deemed to contain the full protein coding sequence were used to produce 413 independent sense lines.
Five T1 developmental phenotypes which were inheritable and co-segregated with the transgene, were obtained. 1.The 'purple vein' phenotype was caused by an antisense Receptor-Like-Kinase construct. Mutants exhibited stress-like anthocyanin pigmentation in the mid-vein and alterations to leaf morphology. 2. A dwarf mutant that was observed in only one line out of 47, and showed no
alteration in targeted mRNA levels, suggesting a dominant T-DNA insertion effect. 3. The 'broomhead' phenotype, characterised by tight bunches of siliques, was caused by overexpression of a translation release factor, eRF1. 4. The 'plentiful' phenotype, characterised by delayed flowering, darker colour and reduced internodal space, was caused by overexpression of a gene of unknown function. 5. The 'sick rosette' phenotype was caused by overexpression of a calcium dependent protein kinase, resulting in dwarfed plants with small twisted rosette leaves.
These mutants will be useful in assigning functions to their corresponding genes, while the entire collection of antisense and sense transgenic lines remains a valuable tool for performing screenings for altered responses to different stimuli; a task already begun in collaboration with other institutions.
A sixth phenotype, 'light green', was found to segregate independently of the T-DNA
and was characterised by a light green leaf blade with green veins (reticulate leaf), similar to previously described Arabidopsis mutants (Kinsman and Pyke, 1998; López- Juez et al., 1998; Rédeiand Hirono, 1964). The ability to complement the most similar mutant, cue1 (Li et al., 1995; Streatfield et al., 1999) suggests that the light green locus (Itg1) is a novel genetic locus required for proper development of the Arabidopsis mesophyll cell layer.