The influence of the plant circadian clock on nodulation in Medicago truncatula

Leguminous plants (or legumes) are well-known for their symbiotic relationship with nitrogen (N) fixing soil bacteria (rhizobia). Through providing a niche to rhizobia in specialized root organelles called nodules and in exchange for plant sugars and nutrients, leguminous plants gain access to the nitrogen fixed by the bacteria. As a result, nodulating plant species can grow in N-depleted soil while most non-legume crop plants require Nfertilizers to reach significant yields. A family of peptides, called Nodule-specific Cysteine- Rich (NCR) peptides, play important roles in rhizobial differentiation and nodulation in certain legumes (e.g., Medicago truncatula) belonging to the Inverted-Repeat Lacking Clade (IRLC). Interestingly, a subset of these NCR gene promoters contain the Evening Element (EE), a cis-regulatory motif which acts as a binding site for a number of plant circadian transcription factors including LATE-ELONGATED HYPOCOTYL (LHY). In order to characterize a potential LHY-NCR-nodulation mechanism, a free-running time-course experiment was carried out where M. truncatula A17 nodules, roots and leaves were sampled over 48 hours. Gene expression analysis through RNA sequencing revealed around 5% of all M. truncatula genes were diurnally oscillating in nodules, including 45 NCRs genes. More than half of these NCRs were highly expressed in the evening period and a significant number of them contain EE motif in their promoters. This supports the hypothesis that these NCRs are negatively regulated by the morning expressed circadian transcription factor, LHY. Furthermore, we have shown that the loss of LHY function leads to reduced nodulation and plant growth. In line with a regulatory role for the clock in nodule metabolic activity, when we clustered different rhythmically expressed plant and rhizobial genes in nodules into four time-of-day groups, we found that modulation of metabolism occurs in waves. This elaborated our understanding of the function of known rhythmic processes (inc. photosynthesis, carbohydrate metabolism) and identified novel functional links. In parallel to the LHY-NCR gene expression study, two cortex-specific GFP expression vectors were prepared and analyzed that could be useful for cell-type specific gene expression in future since the cortex is a site of nodule development. Application of our data is likely to enable us to maximize symbiotic N-fixation and potentially help us to employ such features in non-legume crop plants, bringing considerable benefits.