Discovery and heterologous reconstitution of a plant noncanonical quasi-circadian gene regulatory network

May 13, 2026Cell

Discovery and lab reconstruction of a plant gene network with an unusual daily rhythm

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Abstract

Circadian rhythms were maintained in refrigerated postharvest strawberries despite an arrhythmic circadian clock gene regulatory network.

A noncanonical gene regulatory network (GRN) involving five uncharacterized transcription factors was identified.
This GRN demonstrated the ability to generate circadian rhythms in a heterologous system.
Downstream genes were regulated by this GRN based on various biochemical assays.
Perturbation of the GRN in fruit increased susceptibility to the pathogen Botrytis cinerea.
The findings suggest that rhythm-generating ability is not limited to traditional circadian clocks.

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Circadian rhythms are mainly generated by a gene regulatory network (GRN) constituted by transcription factors (TFs). Comparisons between plant and mammalian circadian clock GRNs suggest conservation of the network architecture rather than its components. Therefore, a rhythm-generating capacity is not restricted to canonical circadian clock GRNs. Here, we showed that although the circadian clock GRN was arrhythmic, circadian rhythms were maintained in refrigerated postharvest strawberries. Through systematic dual-luciferase assays and network analysis, we discovered a noncanonical GRN pillared by five uncharacterized TFs. We developed a heterologous reconstitution system and demonstrated the rhythm-generating ability of this GRN. Subsequent systematic evolution of ligands by exponential enrichment followed by high-throughput sequencing (SELEX-seq), electrophoretic mobility shift assay (EMSA), and DNA affinity purification (DAP)-qPCR analyses suggested that this GRN was responsible for the circadian rhythms of downstream genes. Fruit-specific perturbation of this GRN led to enhanced susceptibility to Botrytis cinerea. Collectively, our study identified a noncanonical quasi-circadian GRN, realized the heterologous reconstitution of eukaryotic circadian GRNs, and demonstrated its function in immune regulation.

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Discovery and heterologous reconstitution of a plant noncanonical quasi-circadian gene regulatory network

Abstract

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