A reactive oxygen species Ca²⁺ signalling pathway identified from a chemical screen for modifiers of sugar‐activated circadian gene expression

Abstract

Sugars are essential metabolites for energy and anabolism that can also act as signals to regulate plant physiology and development. Experimental tools to disrupt major sugar signalling pathways are limited. We performed a chemical screen for modifiers of activation of circadian gene expression by sugars to discover pharmacological tools to investigate and manipulate plant sugar signalling.Using a library of commercially available bioactive compounds, we identified 75 confident hits that modified the response of a circadian luciferase reporter to sucrose in dark-adapted Arabidopsis thaliana seedlings. We validated the transcriptional effect on a subset of the hits and measured their effects on a range of sugar-dependent phenotypes for 13 of these chemicals. Chemicals were identified that appear to influence known and unknown sugar signalling pathways.Pentamidine isethionate was identified as a modifier of a sugar-activated Ca 2+ signal that acts as a calmodulin inhibitor downstream of superoxide in a metabolic signalling pathway affecting circadian rhythms, primary metabolism and plant growth.Our data provide a resource of new experimental tools to manipulate plant sugar signalling and identify novel components of these pathways.

HEXOKINASE-dependent regulation of WRKY transcription factors in Arabidopsis

“…Sugar promotes accumulation of superoxide and cytosolic Ca 2+ in both Arabidopsis and rice and acts to increase expression of circadian clock genes in the evening (Román et al, 2021;Chen et al, 2022;Li et al, 2022). Pharmacological inhibition of this superoxide-Ca 2+ signalling pathway in Arabidopsis lengthens circadian period (Li et al, 2022). We found that the same inhibitors also reduce the response the WRKY transcripts to sucrose (Fig 2), suggesting they are also activated by the superoxide-Ca 2+ pathway.…”

“…Using qRT-PCR, we confirmed that these four genes were upregulated in response to sucrose, compared to mannitol controls in dark-adapted seedlings (Fig 2). Furthermore, we tested whether the induction of these genes by sucrose was inhibited by DPI or PI, two chemicals that inhibit a sugar-activated superoxide-Ca 2+ signalling pathway (Román et al ., 2021; Li et al ., 2022). Upregulation of all four WRKY genes by sugar was significantly inhibited by either DPI or PI (Fig 2A).…”

“…We have proposed that sugar-activated superoxide signalling contributes to growth because DPI and PI both inhibit hypocotyl elongation in dark-grown seedlings (Román et al ., 2021; Li et al ., 2022). We tested the effect of sucrose on hypocotyl growth in the wrky mutants.…”

Galileo Estopare Araguirang

“…Furthermore, Li et al . (2022) recently provided a new strategy and set of pharmacological tools to dissect signalling pathways that regulate plant growth. But most relevant to my current work, I am fascinated by the ongoing debate that wrestles on the polarising and controversial primary function and functional significance of anthocyanins in plants (Renner & Zohner, 2019; Pena‐Novas & Archetti, 2020; Agati et al ., 2021).…”

“…As someone who started in photobiochemistry, I also enjoyed reading the insightful Commentary by Professor Batschauer (2022) on two papers (Liu et al, 2022;Miao et al, 2022) regarding the stability and regulation of cryptochrome 1 in response to high light. Furthermore, Li et al (2022) recently provided a new strategy and set of pharmacological tools to dissect signalling pathways that regulate plant growth. But most relevant to my current work, I am fascinated by the ongoing debate that wrestles on the polarising and controversial primary function and functional significance of anthocyanins in plants (Renner & Zohner, 2019;Pena-Novas & Archetti, 2020;Agati et al, 2021).…”