Using global expression to study tropic growth: A case for auxin mediated changes in transcription and a role for NPH4/ARF7-mediated target activation in tropic responsiveness

The establishment of normal phototropic and gravitropic curvatures in plant stems requires the function of the auxin-responsive transcriptional activator NPH4/ARF7 (Harper et al., 2000; Watahiki et al., 1997; Liscum and Reed 2002). In addition to providing genetic support for the long-held notion that tropic responses require the formation of and response to localized changes in auxin concentration, the dependence of tropic responses on NPH4/ARF7 further suggests that auxin-dependent changes in gene expression are critical to these responses (Harper et al., 2000; Tatematsu et al., 2004). In an attempt to identify "tropic stimulus-induced" (TSI) genes, we used mRNAs derived from phototropically and gravitropically stimulated Brassica oleracea seedlings to probe Arabidopsis thaliana whole genome microarrays (Esmon et al., 2006). Specifically, we isolated mRNA from "lit" and "shaded" stem flanks of unilaterally irradiated Brassica seedlings and "top" and "bottom" stem flanks of seedlings whose growth axis was reoriented 90° to stimulate a gravitropic response (Esmon et al., 2006). Our results indicate that a small suite of TSI genes are differentially expressed between opposing flanks, including two indole-3-acetic acid-amido synthetases (DFL1 and GH3-5), three cell wall-modifying enzymes (EXPa1, EXPa8 and SKS1), two transcription factors (bHLH134 and HAT2) and one gene of unknown function (SAUR50).; Further investigation of the interaction between these TSI genes and NPH4/ARF7 by using chromatin immunoprecipitation (ChIP) assays has lead to preliminary results that suggest NPH4/ARr7 does bind to certain auxin response elements (AuxREs) within the promoters of at least two TSI genes and does so in a preferential manner under auxin and blue-light stimulations. In addition to testing the remaining TSI genes for interaction with NPH4/ARF7, we are generating and screening RNA interference (RNAi) loss-of-function lines for each of the TSI genes to assess their physiological roles in the development of tropic responses.; We have also begun culling the data from our previous microarray experiments in the context of tropism-specific global expression changes. Results from clustering analyses corroborate the results of the comparison rank analyses. We have been able to uncover suites of genes that are both tropically responsive in a stimulation specific manner and are expressed in temporally distinct patterns.