Legumes house nitrogen-fixing endosymbiotic rhizobia in specialized polyploid cells within root nodules, which undergo tightly regulated metabolic activity. By carrying out expression analysis of transcripts over time in Medicago truncatula nodules we found that the circadian clock enables coordinated control of metabolic and regulatory processes linked to nitrogen fixation. This involves the circadian clock-associated transcriptional factor LATE ELONGATED HYPOCOTYL (LHY), with lhy mutants being affected in nodulation. Rhythmic transcripts in root nodules include a subset of Nodule-specific Cysteine Rich peptides (NCRs) that have the LHY-bound conserved Evening Element in their promoters. Until now, studies have suggested that NCRs act to regulate bacteroid differentiation and keep the rhizobial population in check. However, these conclusions came from the study of a few members of this very large gene family that has complex diversified spatio-temporal expression. We suggest that rhythmic expression of NCRs may be important for temporal coordination of bacterial activity with the rhythms of the plant host, in order to ensure optimal symbiosis.

See Achom et al. (2022) Journal of Experimental Botany.

All Supplementary Datasets have full legends within the xlsx files.

Supplementary Dataset S1. Medicago truncatula A17 transcript IDs detected in 40 day-old A17 nodules and information on their oscillating behavior. 

Supplementary Dataset S2. Medicago truncatula A17 transcript IDs detected in 40 day-old A17 roots and information on their oscillating behavior. 

Supplementary Dataset S3. Expression of circadian clock gene transcripts detected in 40 day-old A17 nodules and information on their oscillating behavior.

Supplementary Dataset S4. Pathway and protein term enrichment in oscillating gene clusters. 

Supplementary Dataset S5. Analysis of the lhy-1 and lhy-2 mutants.

Supplementary Dataset S6. Genes annotated as NCRs in Medicago truncatula A17 and information on their expression levels, oscillating behaviour and presence of motifs. 

Supplementary Dataset S7. Motif analysis in NCRs and cycling genes.