Members of the GTP-binding protein superfamily are known to be involved in many essential processes from signal transduction to protein synthesis. The newest type, Developmentally Regulated GTP-binding proteins (DRGs), are found in a diverse range of organisms, and all are highly homologous suggesting a common, fundamental function. An Arabidopsis DRG homologue, AtDRG1, was identified in an expressed sequence tagged (EST) database and was characterised in detail in an effort to resolve the function of these proteins in plants.
Phylogenetic analysis of all DRGs revealed that the family is divided into two groups (A and B) and that each organism is likely to contain two related DRG genes. The AtDR1 sequence was used to screen Arabidopsis databases revealing two DRG homologues: AtDRG2 and AtDRG1b, a duplicate of AtDRG1 which does not appear to be expressed in the Arabidopsis tissues tested.
To study AtDRG1 at the protein level, antibodies were made against recombinant AtDRG1. In plant extracts, these antibodies recognise two proteins which may be either the two Arabidopsis DRGs or two forms of AtDRG1. The AtDRG1 and AtDRG2 genes exhibit similar expression patterns in Arabidopsis tissues, and both are regulated during germination and early growth. Interestingly, mRNA levels do not always correspond to relative protein levels suggesting that these genes are post-transcriptionally regulated. AtDRG2 is strongly induced in response to most stresses and hormones, whereas AtDRG1 expression is either not affected or is repressed. This differential regulation may be an indication that the Arabidopsis DRGs have divergent cellular roles.
Transgenic plants which over- or under-express the AtDRG1 gene were produced, however analysis of the protein levels in these plants revealed that only one sense line had slightly increased AtDRGl1 protein levels, and only four of the lines exhibited significantly reduced protein levels (two antisense and two sense). This is likely a result of the stringent post-transcriptional regulation of DRG proteins. Representatives of the sense and antisense lines grew identically to control lines during all developmental stages from germination to maturity, however the sense line exhibited decreased root growth to a statistically significant degree when exposed to the stress of seedling manipulation. This suggests that either AtDRG1 or its post-transcriptional regulation is responsive to certain stress types.
The sub-cellular location of the Arabidopsis DRG proteins was determined through immunolocalisation experiments in which they appear to be located to elliptical structures present in the cytoplasm and vacuole. Visually these structures are similar to yeast vesicles involved in cytoplasm to vacuole targeting and thus may be involved in similar protein transport processes in plants.
A yeast-two-hybrid screen was conducted with an Arabidopsis cDNA library to identify proteins which interact with AtDRG1. Sixteen putative AtDRG1 interactors were found, although the function of most was unknown. 75% of the AtDRG1 interactors were predicted to be located in cellular compartments which would physically separate them from AtDRG1. This result reinforces the suggestion by the immunolocalisation results that AtDRG1 is involved in protein transport, however confirmation of in planta interaction is required before further hypotheses can be made.
A positive from the yeast-two-hybrid screen, DRGip1, interacts with AtDRG1 in vitro and was found to be expressed only in developing seedlings and roots. This expression pattern differs significantly from that of AtDRG1 which may be an indication that AtDRG1 interacts with different proteins in different tissues and thus may be involved in several cellular pathways. Hormone root length assays of over- and under-expressing DRGip1 transgenic plants suggests that this gene is involved in root growth, possibly through a pathway regulated by jasmonate and cytokinin interactions.
The results presented here have revealed that the DRG family members in Arabidopsis are highly regulated and may be involved in many processes from stress and hormone responses to development. Several results suggest that AtDRG1 is involved in protein transport, and while the precise mechanism remains unknown it may act m a regulatory role or as a plant-wide coordinator of certain cellular processes. This research has provided the first detailed characterisation of a plant DRG and will provide a foundation for further investigations, not only for plant research but for DRG research into all organisms.