Analysis of AtGUS1 and AtGUS2 in Arabidopsis root apex by a highly sensitive TSA-MISH method

A new highly sensitive whole-mount in situ hybridization method, based on tyramide signal amplification (TSA-MISH) was developed and a combined GFP detection and TSA-MISH procedure was applied for the first time in plants, to precisely define the spatial pattern of AtGUS1 and AtGUS2 expression in the root apex. GUS, belonging to GH79 family, are widely distributed in plants but their functional role has been not yet fully investigated. In the model system Arabidopsis thaliana three different AtGUS genes have been identified which encode proteins with putative different fates. Endogenous GUS expression has been detected in different organs and tissues, but the cyto-histological domains of gene expression remain unclear. The results here reported show co-expression of AtGUS1 and AtGUS2 in different functional zones of root apex (the cap central zone, the root cap meristem, the staminal cell niche and the cortical cell layers of proximal meristem), while AtGUS2 is exclusively expressed in the cap peripheral layer and in the epidermis in the elongation zone. Interestingly, both genes are not expressed in the stelar portion of proximal meristem. A spatial (cortex vs stele) and temporal (proximal meristem vs transition zone) regulation of AtGUS1 and AtGUS2 expression is therefore active in the root apex. This expression pattern, although globally consistent with the involvement of GUS activity on both cell proliferation and elongation, clearly indicated that AtGUS1 and AtGUS2 could control downstream distinct process depending on the developmental context and the interaction with other players of root growth control. In the future, the developed new approaches could be very useful to dissect such interactions.