Strigo-D2—a bio-sensor for monitoring spatio-temporal strigolactone signaling patterns in intact plants

Abstract

Strigolactones (SLs) are a class of plant hormones that mediate biotic interactions and modulate developmental programs in response to endogenous and exogenous stimuli. However, a comprehensive view on the spatio-temporal pattern of SL signaling has not been established, and tools for a systematic in planta analysis do not exist. Here, we present Strigo-D2, a genetically encoded ratiometric SL signaling sensor that enables the examination of SL signaling distribution at cellular resolution and is capable of rapid response to altered SL levels in intact Arabidopsis (Arabidopsis thaliana) plants. By monitoring the abundance of a truncated and fluorescently labeled SUPPRESSOR OF MAX2 1-LIKE 6 (SMXL6) protein, a proteolytic target of the SL signaling machinery, we show that all cell types investigated have the capacity to respond to changes in SL levels but with very different dynamics. In particular, SL signaling is pronounced in vascular cells but low in guard cells and the meristematic region of the root. We also show that other hormones leave Strigo-D2 activity unchanged, indicating that initial SL signaling steps work in isolation from other hormonal signaling pathways. The specificity and spatio-temporal resolution of Strigo-D2 underline the value of the sensor for monitoring SL signaling in a broad range of biological contexts with highly instructive analytical depth.

Rapid analysis of strigolactone receptor activity in a Nicotiana benthamiana dwarf14 mutant

“…An Arabidopsis line expressing a D14 fusion to luciferase has been developed as an in vivo assay for the ability of various SLs and SL analogs to induce D14 degradation (Sanchez et al, 2018 ). Most similar to our system are the ratiometric SL signaling sensors StrigoQuant and Strigo‐D2 (Samodelov et al, 2016 ; Song et al, 2022 ). StrigoQuant expresses Renilla luciferase and a SMXL6 fusion to firefly luciferase in a single transcript.…”

“…Coexpression of rice D14 with Strigoquant is able to restore SL‐induced SMXL6‐degradation responses to the Arabidopsis d14 mutant, demonstrating that it is feasible to test SL receptor variants with this system (Samodelov et al, 2016 ). Strigo‐D2 coexpresses from separate 35S promoters a SMXL6 C‐terminal domain (SMXL6‐D2) fused to the yellow fluorescent protein mVenus and a nuclear‐localized mCherry reference protein (Song et al, 2022 ). Overexpression of SMXL6‐D2‐mVenus has less negative impact on Arabidopsis growth and produces higher signal intensities than full‐length SMXL6‐mVenus.…”

“…The Strigo‐D2 system provides a rapid (within 20 min) and sensitive readout of SL response in transgenic Arabidopsis plants. Responses to applied 5‐deoxystrigol concentrations as low as 5 nM can be detected, and a range of responsiveness can be observed in different plant tissues (Song et al, 2022 ). Putatively, the activity of D14 variants could be evaluated in the d14 Strigo‐D2 background through the generation of stable transgenic lines, although this may be time‐consuming.…”

“…mutant, demonstrating that it is feasible to test SL receptor variants with this system (Samodelov et al, 2016). Strigo-D2 coexpresses from separate 35S promoters a SMXL6 C-terminal domain (SMXL6-D2) fused to the yellow fluorescent protein mVenus and a nuclear-localized mCherry reference protein (Song et al, 2022).…”

How Strigolactone Shapes Shoot Architecture

“…Interestingly, several SL-sensing genes ( D14 , MAX2 , SMAXL6 , SMAXL7 , and SMAXL8 ) share the expression pattern along the vascular strands with SL-biosynthetic genes ( Figure 4 ; Gao et al, 2004 ; Shen et al, 2007 ; Stirnberg et al, 2007 ; Chevalier et al, 2014 ; Soundappan et al, 2015 ; Song et al, 2022 ). In general, the identity of the cells that express SL-sensing genes along the vasculature is uncertain; however, for the SL receptor D14, expression was attributed to the phloem in Arabidopsis roots ( Chevalier et al, 2014 ), and in the axillary buds of rice ( Kameoka et al, 2016 ), suggesting that SL perception may be possible in these tissues.…”