These authors contributed equally to this work.
Functional characterization of the CKRC1/TAA1 gene and dissection of hormonal actions in the Arabidopsis root
Version of Record online: 1 MAR 2011
© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd
The Plant Journal
Volume 66, Issue 3, pages 516–527, May 2011
How to Cite
Zhou, Z.-Y., Zhang, C.-G., Wu, L., Zhang, C.-G., Chai, J., Wang, M., Jha, A., Jia, P.-F., Cui, S.-J., Yang, M., Chen, R. and Guo, G.-Q. (2011), Functional characterization of the CKRC1/TAA1 gene and dissection of hormonal actions in the Arabidopsis root. The Plant Journal, 66: 516–527. doi: 10.1111/j.1365-313X.2011.04509.x
- Issue online: 26 APR 2011
- Version of Record online: 1 MAR 2011
- Accepted manuscript online: 22 JAN 2011 07:43AM EST
- Received 6 May 2010; revised 12 January 2011; accepted 19 January 2011.
Figure S1. Root phenotype responses to different hormones/chemicals in WT and the mutant as compared to those on MS medium (a), showing the curling phenotypes of the mutant induced by CKs (b–d) and TIBA (e), and the rescuing effects of auxins (f–h) and ACC (weak, i).
Figure S2.ckrc1-1 mutant phenotypes. (a) 3-day old dark-grown WT (left) and ckrc1-1 mutant (right) seedlings. ckrc1-1 mutant plants developed shorter hypocotyls with normal apical hook. (b) ckrc1-1 mutant plants exhibit late flowering phenotype compared with WT plants grown under the same condition and normal GR of inflorescence stems in soil-grown plants (c) or hypocotyls of etiolated seedlings (d).
Figure S3. Reduced expression of CYCB1 genes in ckrc1-1 root tips by qRT-PCR, Shown are means ± SDs of three separate biological repeats. Student’s t-test, *P < 0.05).
Figure S4. Primary root elongation response of ckrc1-1 mutant. WT and ckrc1-1 mutant were grown in presence of various concentrations of 6-BA (a), TDZ (b), 1-NAA (c) and 2,4-D (d) and primary root elongation was measured in 7 days. All experiments were repeated three times. Shown are means ± SDs with n = 35–60 in each repeat.
Figure S5. Rescuing CK-induced root curling in ckrc1-1mutant. WT (left) and ckrc1-1 mutant (right) plants were grown on vertically oriented plates of MS (a) or MS supplemented with 0.1 μM ZT (b), 0.32 μM ACC and 0.1 μM ZT (c), 0.01 μM IAA and 0.1 μM ZT (d), 0.1 μM NAA and 0.1 μM ZT (e), or 0.01 μM 2,4-D and 0.1 μM ZT (f) for 7 days.
Figure S6. Rescuing root GR defects in ckrc1-1 mutant by auxin. (a) 10 day-old ckrc1-1 seedlings grown vertically on plates of MS or MS supplemented with IAA, NAA, 2,4-D or ACC at the indicated concentrations were reoriented 90° for 48 h. (b) CKs do not affect that response in WT at their concentrations with 50% growth inhibition on roots.
Figure S7. Allelic series tests on MS media with 0.1 μM ZT. Compared with WT (a), ckrc1-1−/− (b) ckrc1-2−/− (d) and ckrc1-3 (f) display similar root curling phenotypes. F1 plants from a cross between WT and ckrc1-1−/− exhibit a WT-like phenotype (c), consistent with the recessive nature of ckrc1-1. On the other hand, F1 plants from crosses between ckrc1-1−/− and ckrc1-2−/− (e), and ckrc1-1−/− and ckrc1-3−/− (g) exhibit no complementation mutant phenotype, indicating that they are allelic mutants.
Figure S8. Root GR defects of ckrc1 and its allelic mutants. (a) Images of 7 day-old seedlings rotated horizontally for 48 h. (b) Quantification of root GR. Root tip curvatures of the majority of WT plants range between 60 and 90° from the initial angle, whereas the root tip curvatures of the ckrc1 mutants range from 0 to 360° from the initial angle. n = 20–50.
Figure S9. Protein sequence alignment of CKRC1 and its homologs (top), highlighting domains of amino acid sequence conservation. Shown in the Bottom panel were phylogenetic relationships using neighbor-joining program.
Figure S10. Phenotypic rescue of ckrc1-1 by exogenous IPA but not L-Trp treatments. WT (left) and ckrc1-1 mutant (right) plants were grown on vertically oriented plates of MS (a) or MS supplemented with 0.1 μM IPA (b), 3 μM L-Trp (c), 0.1 μM ZT (d), 0.1 μM IPA and 0.1 μM ZT (e), or 3 μM L-Trp and 0.1 μM ZT (f) for 7 days.
Figure S11. Comparison of the responses to exogenous IAA and ZT treatments of IAA1/IAA2 and ARR5/ARR15 genes expression between WT and ckrc1-1 by qRT-PCR. Shown are means ± SDs of three separate biological repeats. Student’s t-test, P < 0.0001).
Figure S12. Effects of ZT and ACC treatments on the expressions of genes related to IAA biosynthesis. Their loss of function (LOF) mutations led to low auxin levels, except Sur1 and Sur2, of which LOF mutations led to high auxin (Woodward & Bartel, 2005; Cheng et al. 2006). Data retrieved from the AtGenExpress Visualization Tool (AVT, http://jsp.weigelworld.org/expviz/expviz.jsp).
Figure S13. Dr5::GUS responses in root tips of 7 d old seedlings grown on MS media with different hormones/chemicals (IAA, 0.04 μM; ZT, 0.1 μM; ACC, 0.32 μM; Ag\, 10 μM). ZT significantly reduced DR5::GUS expression in the ckrc1-1 mutant but not the WT root-tips (a), whereas 0.32 μM ACC appeared to have weak positive effect, especially in the central cylinder above QC (b). n = 20–30.
Figure S14. Effects of auxin, CK and ethylene on asymmetric DR5rev::GFP expression in root tips of ckrc1-1 mutant plants upon gravistimulation. Four-day-old seedlings grown on vertically oriented plates were transferred to new plates supplemented with 0.01 μM IAA, 0.32 μM ACC, or 0.1 μM ZT and reoriented 90° for 3–4 h. Shown were confocal images of DR5rev::GFP expression in WT (left two panels) and ckrc1-1 mutant (right two panels). Red fluorescence of PI staining indicates cell wall. n = 20–30.
Appendix S1. T-DNA flanking sequence cloned by I-PCR in ckrc1-1.
Appendix S2. PCR primer sequences.
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