Crosstalk between ethylene, light, and brassinosteroid signaling in the control of apical hook formation

Most dicotyledonous seedlings form an apical hook when seeds germinate in darkness (such as when buried in soil) that protects the cotyledons and apical meristem as the hypocotyl elongates through the soil toward the surface. The formation of the apical hook involves rapid and asymmetrical growth directed by auxin and Small Auxin UP RNA (SAUR) genes. Previous reports indicate that SAUR17 is specifically expressed in the apical part of dark-grown seedlings (Wang et al. 2020). At least two known pathways influence SAUR17 gene expression during apical hook formation: one related to light/dark responses and the second dependent upon ethylene, the biosynthesis of which is induced by mechanical pressure during germination and hypocotyl elongation through the soil.

In this issue of The Plant Cell, Jiajun Wang and colleagues (Wang et al. 2022) report the participation of brassinosteroid (BR) signaling and crosstalk between the BR and both the light/dark and ethylene response pathways in the control of SAUR17 expression and apical hook formation after germination. First, the authors analyzed the relationship between light, ethylene, and auxin signaling pathways in regulating SAUR17 gene expression in young dark-grown seedlings. The authors found that light inhibits while PHYTOCHROME-INTERACTING FACTOR3 (PIF3) and PIF4 induce SAUR17 expression, with the highest SAUR17 expression coinciding with the PIF3 and PIF4 proteins abundance peak. Constitutive expression of SAUR17 in the pif1 pif3 pif4 pif5 quadruple mutant (pifq) restored the WT cotyledon phenotype (see Figure). In the case of ethylene, the saur17 mutant is insensitive to the ethylene-induced apical hook formation (Wang et al 2020); additionally, the application of 1-Aminocyclopropane-1-carboxylic acid (the immediate ethylene precursor) to dark-grown seedlings increased the expression of SAUR17. ETHYLENE INSENSITIVE3 (EIN3) and EIN3-like (EIL1) are critical mediators of ethylene-mediated SAUR17 expression. However, SAUR17 expression does not change in response to exogenous auxin treatments as do other SAUR genes, such as SAUR15.

Figure.

BR integration in pathways controlling apical hook formation. A, Constitutive expression of SAUR17 suppressed the cotyledon-opening phenotype of pifq in the dark. Phenotypes of the dark-grown pifq mutant and three independent transgenic lines of Enh35S:ProSAUR17:SAUR17-GFP/pifq compared with wild-type (Col). B, A model illustrating the effect of BR in promoting the etiolated development of apical organs in the darkness. BR inhibits the transcription of EBF1/2 via BZR1, resulting in stabilization of the EIN3/EIL1 and PIF3 proteins. Adapted from Wang et al. (2022), Figures 1 and 7.

Previous reports indicate that BR signaling is necessary for etiolation (Li et al., 1996). Plants disrupted in BR biosynthesis or signaling lack the apical hook and display separate cotyledons and short hypocotyls. The application of eBL (2,4-epibrassinolide, a BR analog) caused enhanced apical hook formation, a response that was compromised in the saur17 mutant. Additionally, BR treatment induced the expression of SAUR17, while the application of brassinazole or propiconazole (inhibitors of BR biosynthesis) caused a marked decline in SAUR17 expression. Other mutants of the BR signaling or biosynthesis pathway (cpd, det2-1, br6ox2) also showed lower SAUR17 expression; additionally, mutants affected in BR transcriptional responses (reduced expression of BZR1 transcription factors) also display reduced hook curvature.

Some hints about the direct control of SAUR17 expression by light (via PIFs), ethylene (via EIN3/EIL1), or BR (via BZR1) came from the analysis of the promoter region. promSAUR17 contains one PBE-box (PIF-binding) and three putative EIN3-binding sites (EBS). Chromatin immunoprecipitation experiments demonstrated that PIFs and BZR1 could bind the PBE-box, while EIN3, PIFs, and BZR1 can bind the EBS. Similar protein–DNA interaction tests were conducted in mutant backgrounds (ein3/eil1 or pifq) that showed an additional level of BR pathway crosstalk with ethylene or light. BZR1 binding to the EBS was abolished in the absence of EIN3/EIL1, while BZR1 displayed lower binding to the PBE-box in the absence of PIF or EIN3/EIL1. BZR1 can bind the SAUR17 promoter in the pifq or ein3/eil1 background, but SAUR17 was not expressed. The pathways are also interconnected at the protein–protein interaction level since PIF3–EIN3 (Liu et al., 2017; Zhang et al., 2018), BZR1–PIF4 (Oh et al., 2012), and BZR1–PIF3 and EIN3–BZR1/BES1 physically interact. The EIN3–BZR1 interaction enables BZR1 to associate with the EBS of the promoter. The dependence of BZR1 for PIFs and EIN3/EIL1 to promote SAUR17 expression is also observed. SAUR17 expression was diminished in pifq, ein3 eil1, and pifq ein3 eil1 mutants. The ein3 eil1 mutant was insensitive to BR treatments. BZR1-induced SAUR17 expression depends on PIFs, EIN3/EIL1, and BR that increases promoter binding of PIFs and EIN3. In summary, EIN3/EIL1 and PIFs play essential roles in BR-induced SAUR17 activation.

Interestingly, BR treatment also affected EIN3 and PIF protein abundance. EIN3 and PIF abundance increased after treatment with eBL and decreased after treatment with brassinazole, a BR biosynthesis inhibitor. Then, the authors focused on EBF1 and EBF2, the ubiquitin ligase components involved in the degradation of EIN3 and PIF3. The expression of EBF genes is affected by BR treatments (eBL), and this effect likely involves BZR1 since there are BZR1-binding sites in the EBF1 and EBF2 promoters; additionally, the transcriptomic analysis indicated that EBF1 and EBF2 are BZR1-repressed genes. EBF1 or EBF2 overexpression eliminates the BR-induced increase of the EIN3 or PIF3 protein abundance, and SAUR17 expression was diminished. Thus, the EBFs are critical factors in BR-induced protein accumulation of EIN3 and PIF3.

In summary, this work describes a multilevel integration of the BR-response pathway in the control of the hook formation in the seedling, involving a BR-triggered decline in EBF expression, resulting in the accumulation of EIN3 and PIF3, causing the enhanced expression of SAUR17. The authors propose that BR affects apical hook development primarily by modulating the light/dark and ethylene pathways.