Plant circadian clock control of Medicago truncatula nodulation via regulation of nodule cysteine-rich peptides

Abstract

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 transcription 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.

Keywords: Medicago truncatula; Circadian biology; nitrogen fixation; nodulation; plant–environment interaction; symbiosis.

Fig. 1.

Oscillating expression of the belowground tissue circadian clocks. (A) Experimental design for 48h time course with sampling points. (B) Nodule and (C) root normalized mean transcription levels of clusters of rhythmic transcripts that peak at different times of the day (gold indicates a morning peak, red an evening peak, and blue a peak at night). Enriched biological pathways in each cluster, protein motifs, and GO terms have been listed in the coloured boxes; see Dataset S4 at Dryad for process details. Arrows indicate the mean MetaCycle predicted peak of transcripts associated with these processes. Black and red symbols indicate the peak transcription of a rhythmically expressed NCR transcript; a red circle indicates that the NCR gene promoter contains both an EE and EER, a triangle indicates that it contains an EE, an inverted triangle that it contains an EER, and a black square indicates that it has neither motif. (D) Expression profiles of lhy (gold) and toc1 (navy blue) in nodules (solid line; see Dataset S1 at Dryad) and roots (dotted line; see Dataset S2 at Dryad). Arrows indicate the time of the peak as predicted by MetaCycle.

Fig. 2.

Loss of M. truncatula LHY expression affects plant rhythmicity and nodulation. (A) Phylogenetic analysis of CCA1/LHY homologues in Plantae and Chlorophyte. MtLHY (Medtr7g118330) shares 36.6% identity at the amino acid level with AtCCA1 (At2g46830) and 44.2% identity at the amino acid level with AtLHY (At1g01060); Aha, Arabidopsis helleri; Aly, Arabidopsis lyrata; At, Arabidopis thaliana; Bdi, Brachypodium distachyon; Bol, Brassica oleracea; Bra, Brassica rapa; Csa, Castanea sativa; Gma, Glycine max; Mcr, Mesembryanthemum crystallinum; Mt, Medicago truncatula; Os, Oryza sativa; Ot, Ostreococcus tauri; Pni, Populus nigra; Pvu, Phaseolus vulgaris; Zm, Zea mays; see Dataset S5 at Dryad for gene IDs. (B) Location of lhy-1 and lhy-2 insertions in the LHY gene. (C) Relative expression of MtLHY in wild-type R108 (green), lhy-1 (orange), and lhy-2 (red) mutant plant leaves in the morning and evening periods. (D) Period of leaf movement rhythms for each genotype inferred from experimental data using the FFT-NLLS algorithm in BioDare2; for data see Dataset S5 at Dryad; black circles indicate individual plants, the diamond indicates mean period. (E) Disrupted leaf movement rhythms in lhy mutants in constant light; dashed vertical lines indicate the mean period for each genotype.

Fig. 3.

Loss of M. truncatula LHY expression affects nodulation under 16L:8D cycles. (A) Plants have a similar dry aboveground weight phenotype in the absence of rhizobial inoculation, but with inoculation the lhy mutants have reduced dry weight; boxplots with individual replicate data; n=24; ∗∗∗P<0.005. (B) Reduced nodule weight for lhy-1 and lhy-2 compared with the wild type R108; n=24; ∗P<0.05. (C) Images of 6-week-old mock- (top row) or Sinorhizobium meliloti WSM1022-inoculated plants grown in perlite–vermiculate pots showing reduced growth (second row) and less ramified nodules in the lhy mutants (lower rows). Scale bars for the upper two rows=10cm, middle row 1cm, lower row 0.1cm. (D) Nodule and nodule meristem lobe counts from inoculated plants; n=21–29; ∗P<0.05, ∗∗∗P<0.005; see Dataset S5 at Dryad for all values and analyses.

Fig. 4.

The evening element is enriched in the promoters of oscillating NCRs. (A) Motif comparison of the A. thaliana LHY/CCA1-binding site (EE) and M. truncatula evening element-related (EER) putative motif. (B) Proportion of 500 bp promoters containing matches to either the EE, EER, or both motifs. (C) Expression profiles of circadian clock genes (black, black dashed line in the graphs) and NCR genes (coloured, coloured solid lines in the graph) within each cluster. The average and range of each group of genes are indicated with lines and a cloud, respectively. NCRs with the EE motif in their promoters are indicated in bold, EER in italic, and both motifs in bold and italic. See Datasets S6 and S7 at Dryad for all values and analyses.