1. Transcriptional Regulation during Gravitropism in Arabidopsis Root Tips Heike Winter Sederoff, Jeffery M. Kimbrough, Raul Salinas-Mondragon and Christopher Brown NSCORT-Plant Gravitational Genomics Group, Dept. Botany and Kenan Institute, NC State University heike_winter@ncsu.edu NC State University Abstract Rapid, temporal changes in the abundance of specific transcripts occur in response to gravity-stimulation and these transcript level changes reveal clusters of coordinating signaling events that are required for the tissue to make a commitment to differential growth. We have carried out microarray analysis of Arabidopsis root tips after gravistimulation (reorientation) and mechanical stress and monitored transcript levels ca. 24,000 genes during the first hour after either stimuli. We identified genes responding to either stimulus specifically as well as genes shared by both signalling pathways. The temporal pattern of transcription and transcript stability of those induced genes are very distinct. We identified stress (gravity vs mechanical) specific gene expression and isoform specific expression. The earliest changes of transcript levels were seen already 2 min after the stimulus. Table I: Summary of transcript levels responding to gravity and/or mechanical stimulation by increased (up) or decreased (down) steady- state levels of transcripts.Gravity 0 2 5 15 30 60 min after reorientation The Approach We are using the Arabidopsis root tip (7 day old dark-grown seedlings) to analyze the changes in transcript levels in response to gravistimulation using the Arabidopsis ATH1 GeneChip (Affymetrix), containing more than 22,500 probe sets representing approximately 24,000 genes. For our time-course experiment, approximately 150 root tips were harvested before (0 time point) and 2, 5, 15, 30, 60 min after reorientation (135◦) by pouring RNAlater (Ambion) onto the plates and cutting off the root tips (5 mm). Mechanical stress controls seedlings (0, 2, 5, 15, 30, 60 min) were moved horizontally for 5 sec without changing their orientation towards the vector of gravity. Methods: Total RNA was extracted and purified over a Qiagen RNeasy column, amplified, and labeled with biotin. 10 ug of biotin labeled aRNA (amplified RNA) was hybridized to the GeneChip® Arabidopsis ATH1 Genome Array (Affymetrix, Santa Clara, CA) containing more than 22,500 probe sets. Figure 1. Scatter Plot: X-axis values (log 10 signal intensities) correspond to transcript profile before gravity stimulation. Y-axis values correspond to transcript profile of root tips harvested after a 2 minute reorientation (135◦) in the gravity vector plane. Innermost parallel green lines flanking the scatter indicate boundary for genes up or down regulated greater than 2 fold (yellow: not expressed, blue: expressed under one condition; red: expressed genes). Each dot represents a unique gene. First Results We are currently still analyzing the vast amount of data generated by this method. To get an impression of the quality of our microarrays, we carried out real-time PCR for eight genes shown to be up-regulated during gravitropism (see Fig. 3). Scatter Plots (see Fig.1) showed excellent correlation in overall gene expression between treatments. Clusters of temporal expression Preliminary results show that different clusters of genes exhibit changes in mRNA abundance with specific patterns following the time-course during the first hour after gravistimulation (Fig.2). Figure 2: Temporal changes of transcript levels of a xyloglucan endotransglycosylase XET (At4g30270), sucrose transporter SUC2, and a lipid- transfer protein-like gene (At2g18370) in Arabidopsis root tips after gravity-stimulation. Figure 3: Decrease of transcript levels of an extensinA (CAA47807.1), RNA binding protein-like (gi:16974555), and pectinesterase-like (PATX:E31214555) in Arabidopsis root tips after gravity- stimulation. Isoform-specific and gravity-specific transcripts For some clusters of genes, we found very specific expression of individual isoforms of gene families to expressed within less than 5 min after gravistimulation. Calmodulin1 (CaM1) was upregulated within 2-5 min after gravistimulation, while none of the other calmodulin isoforms showed significant effects (Fig. 5). After mechano-stimulation, CaM1 transcript levels were down-regulated (Fig.4). Similar kinetics and specificity was found for the mu isoform of 14-3-3’s. Figure 5: Specific isoforms of CaM and 14-3-3 respond to gravistimulation. Upregulation of CaM1 and 14-3-3 mu, occurred within less than 5 min and steady-state transcript levels were back to control levels after 30 min. Figure 4: Transcript levels of CaM1 in Arabidopsis root tips after gravistimulation compared to transient mechanical stress. Data from microarray analysis (n=2) and confirmed by real-time PCR (with Actin8 as reference). Upregulation of CaM1 occurs within 5 min of gravistimulation. Acknowledgements: This work was supported by NASA. We thank Nina Allen, Wendy Boss, Eric Davies, Steve Huber, Gloria Muday, Imara Perera, Dominique Robertson, and Ron Sederoff for stimulating discussion and support. Figure 6: Hierarchical cluster showing mechanical stress regulated transcripts For specific transcripts, changes in the rates of transcription vs transcript degradation lead to decreases of their steady- state levels (Fig. 3). Mechanical 0 2 5 15 30 60 min after reorientation We found about 1,563 genes were regulated by gravity. Of those, 1,414 showed significant increases in transcript abundance during gravistimulation while 149 showed decreased levels. During mechano- stimulation, 1,350 genes showed significant regulation, 1,115 were increased, while 235 showed a decrease in transcript abundance. Gravity-specific changes were detected for 1,168 transcripts, while 956 transcript showed changes in transcript abundance only after mechano- stimulation. Especially interesting are those genes which show opposite responses in both signaling networks (see Table I). Stress:updowntotal Gravity 14141491563 Mechanical 11152351350 Gravity (specific)10581101168 Mechanical (specific)788168956 Both Stresses similar response:32448397 opposite response: 25 Figure 7: Hierarchical cluster showing gravity regulated transcripts