Regulation of plant phototropic growth by NPH3/RPT2-like substrate phosphorylation and 14-3-3 binding

Stuart Sullivan,Thomas Waksman,John M Christie,Noriyuki Suetsugu,Louise Henderson,Dimitra Paliogianni,Melanie Lütkemeyer

doi:10.1038/s41467-021-26333-5

Abstract

Polarity underlies all directional growth responses in plants including growth towards the light (phototropism). The plasma-membrane associated protein, NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key determinant of phototropic growth which is regulated by phototropin (phot) AGC kinases. Here we demonstrate that NPH3 is directly phosphorylated by phot1 within a conserved C-terminal consensus sequence (RxS) that is necessary to promote phototropism and petiole positioning in Arabidopsis. RxS phosphorylation also triggers 14-3-3 binding combined with changes in NPH3 phosphorylation and localisation status. Mutants of NPH3 that are unable to bind or constitutively bind 14-3-3 s show compromised functionality consistent with a model where phototropic curvature is established by signalling outputs arising from a gradient of NPH3 RxS phosphorylation across the stem. Our findings therefore establish that NPH3/RPT2-Like (NRL) proteins are phosphorylation targets for plant AGC kinases. Moreover, RxS phosphorylation is conserved in other members of the NRL family, suggesting a common mechanism of regulating plant growth to the prevailing light environment.

Highlights

Polarity underlies all directional growth responses in plants including growth towards the light
In order to identify additional components involved in blue light signalling, GFPNPH3 was immunoprecipitated from etiolated nph[3] mutant seedlings expressing functional NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3)::GFP-NPH328
Anti-GFP immunoprecipitations (IPs) were performed on total protein extracts from seedlings maintained in darkness or after a brief blue light treatment (20 μmol m−2 s−1 for 15 min) to capture early signalling events

Summary

Introduction

Polarity underlies all directional growth responses in plants including growth towards the light (phototropism). The plasma-membrane associated protein, NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key determinant of phototropic growth which is regulated by phototropin (phot) AGC kinases. We demonstrate that NPH3 is directly phosphorylated by phot[1] within a conserved C-terminal consensus sequence (RxS) that is necessary to promote phototropism and petiole positioning in Arabidopsis. RxS phosphorylation is conserved in other members of the NRL family, suggesting a common mechanism of regulating plant growth to the prevailing light environment. Phototropism is induced by UV/blue light and is mediated by two phototropin (phot) light-activated kinases, phot[1] and phot[22]. BLUE LIGHT SIGNALLING 1 (BLUS1) and CONVERGENCE OF BLUE LIGHT AND CO2 1 (CBC1) are phot1-kinase substrates involved in blue light-induced stomatal opening[15,16], while phosphorylation of ATP- BINDING CAS-. Given the variety of physiological responses mediated by phot signalling, further phot kinase substrates likely await identification[20]

Methods

Results

Conclusion