Jun-Li Wang, Ming Wang, Li Zhang, You-Xia Li, Jing-Jing Li, Yu-Yang Li, Zuo-Xian Pu, Dan-Yang Li, Xing-Nan Liu,

Wang Guo, Dong-Wei Di, Xiao-Feng Li, Guang-Qin Guo, and Lei Wu

PNAS; April 18, 2024; 121 (17) e2314353121

Significance

The apical hook protects cotyledons and shoot apical meristem from injuries during seedling emergence from the soil. Auxin plays a key regulatory role in hook formation. Recently, a noncanonical auxin signaling pathway was discovered during apical hook development. Asymmetrical accumulation of auxin in the hook on its concave side triggers cleavage of the C-terminal of TMK1, which then travels to the nucleus and stabilizes two noncanonical Aux/IAAs (IAA32/34) by phosphorylation. Phosphorylated IAA32/34 inhibit elongation of cells on the concave side, resulting in hook formation. This work shows that IAA32/34 degradation is initiated through ubiquitination by WAV E3 ligases, which is inhibited by TMK1-mediated phosphorylation. Our studies identified components critical for IAA32/34 turnover in this TMK1-mediated auxin signaling pathway.

Abstract

Auxin regulates plant growth and development through downstream signaling pathways, including the best-known SCF^TIR1/AFB-Aux/IAA-ARF pathway and several other less characterized “noncanonical” pathways. Recently, one SCF^TIR1/AFB-independent noncanonical pathway, mediated by Transmembrane Kinase 1 (TMK1), was discovered through the analyses of its functions in Arabidopsis apical hook development. Asymmetric accumulation of auxin on the concave side of the apical hook triggers DAR1-catalyzed release of the C-terminal of TMK1, which migrates into the nucleus, where it phosphorylates and stabilizes IAA32/34 to inhibit cell elongation, which is essential for full apical hook formation. However, the molecular factors mediating IAA32/34 degradation have not been identified. Here, we show that proteins in the CYTOKININ INDUCED ROOT WAVING 1 (CKRW1)/WAVY GROWTH 3 (WAV3) subfamily act as E3 ubiquitin ligases to target IAA32/34 for ubiquitination and degradation, which is inhibited by TMK1c-mediated phosphorylation. This antagonistic interaction between TMK1c and CKRW1/WAV3 subfamily E3 ubiquitin ligases regulates IAA32/34 levels to control differential cell elongation along opposite sides of the apical hook.

See: https://www.pnas.org/doi/10.1073/pnas.2314353121

Figure 1: Loss of WAV3 E3 ubiquitin ligase activity is associated with apical hook defect. (A) Diagrams of the WAV3 protein and its truncated version. The conserved amino acids in its RING domain are shown at the Bottom. (B) Detected ubiquitin ligase activity of the truncated WAV3 in vitro. (C) C125 is essential for ubiquitin ligase activity of the WAV3 truncations. GST was used as negative control, while Coomassie Brilliant Blue (CBB) staining was the loading control. Comparable amount of GST-WAV3N2, GST-WAV3N2^H147Y, and GST-WAV3N2^C125S is shown in the GST-blot in SI Appendix, Fig. S4. (D) Representative apical hook phenotypes of the denoted genotypes/transgenic complementation lines 48 h after germination. Quantification of WAV3 overexpression levels is shown in SI Appendix, Fig. S5B. (E) Quantification of apical hook curvature (angle α, n ≥ 20). The box plot shows the first and third quartiles, split by the median and extended to minimum and maximum values. Different letters indicate significant differences at P < 0.05 according to one-way ANOVA with Tukey’s multiple comparisons test.