BioProject

Wild tobacco flowers wave rhythmically to facilitate specific pollinator interactions. This movement behavior is controlled by a regulatory network that involves the circadian clock- and auxin-signaling pathways. The plant hormone auxin, similarly to its function in tropic movements, acts as growth regulator in the circadian regulation of floral movement. Dorsoventral asymmetry in auxin levels and auxin transcriptional responses mediate the growth responses in the floral peduncle that make flowers move. Multiple components of the auxin-signaling pathway and auxin responses are under the control of circadian clock. However, it is unclear where these two pathways intersect and how collectively contribute to regulate specific rhythmic outputs. Here we found that the blue light photoreceptor and circadian clock component ZEITLUPE (ZTL) controls auxin responses through the regulation of the auxin-signaling pathway in a time-of-day and blue light specific manner. Abrogation of ZTL expression abolishes flower movement and the temporal gating of auxin-induced growth responses in the floral peduncle. ZTL regulates transcription and directly interacts with indole-3-acetic acid inducible 19 (IAA19), a circadian controlled gene that regulates development of curvature in moving organs. Indicating that ZTL modulates auxin sensitivity in part through the regulation of AUX/IAA transcriptional repressors. At night, growth responses in the peduncle to the synthetic auxin 2,4-D revealed that ZTL additionally controls auxin responses regulating auxin homeostasis. These results indicate that ZTL conveys temporal and environmental information, at multiple levels, into the auxin signaling-pathway and in this way sculpts the temporal gating of auxin responses that allow flowers to move. To gain further insight into the molecular basis of temporal regulation of the movement of flowers we used a whole genome microarray as a discovery platform to identify genes differentially expressed in a RNAi knockdown line silenced in the expression of the circadian clock component ZEITLUPE (irZTL-314). Overall design: Peduncles of empty vector (EV) and the RNAi knockdown irZTL were plants collected at ZT0. Each replicate is a pool of peduncles collected at same timepoint. Three replicate samples per genotype were used for the analysis. Total RNA was extracted from floral peduncles using the Plant RNeasy kit (Qiagen) and quality checked by spectrophotometry (NanoDrop). Genomic DNA was removed by DNAse treatment (Ambion) following manufacturer instructions. RNA was cleaned up with RNeasy MinElute columns (Qiagen) and the RNA quality was checked with the RNA 6000 Nano kit (Agilent) in the Agilent 2100 Bioanalyzer (Agilent). RNA was labeled with Cyanine-3 (Cy3) with the Quick Amp labeling kit (Agilent) according to manufacturer instructions and followed by RNAeasy column purification (Qiagen). Dye incorporation and cRNA yield were checked with a NanoDrop ND-1000 Spectrophotometer. Samples were hybridized in a N. attenuata whole genome single color array (Agilent 8×60K; GPL19764). Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565BA) using one color scan setting. Probe feature extraction was performed with the feature extraction software (Agilent). Quality control, data filtering and normalization were performed in Genespring GX (Agilent). Probe filtering was performed in Genespring GX using the platform flag (gIswellAboveBG). Probes that did not pass the condition in all replicates for one of the conditions were filtered out. Probe signals were log2 transformed and normalized to the 75th percentile. Differential expression analysis between peduncles of EV and irZTL-314 plants at ZT0 was performed with a t-test (fold change>2, padj<0.05) in Genespring GX (Agilent). Probe to gene association and gene annotations were based in the N. attenuata genome assembly v2.5.