Adapting the Lateral Root-Inducible System to Medicago truncatula.

Almost all legume plants have the capacity to form two types of root organs: lateral roots and nodules (that will host rhizobia that fix nitrogen). Transcriptomic analyses are useful to understand both the similarities and differences between nodule and LR formation and to compare the LR developmental programs used by Arabidopsis and model legumes such as Medicago truncatula. However, in M. truncatula as in Arabidopsis, root cells "committed" to LR formation programs are scattered along the primary root and localized in the inner most layers of the root. To gain access to these cells, a lateral root-inducible system (LRIS) was first developed in Arabidopsis. This LRIS was recently shown to be effective in maize as well. Here we present a LRIS protocol adapted to the model legume Medicago truncatula. Using the same auxin transporter inhibitor and permeant auxin molecules used for Arabidopsis and maize but with slight modifications in their concentrations, we obtained very efficient enrichment and synchronization in LR development stages in M. truncatula.

Nod factors potentiate auxin signaling for transcriptional regulation and lateral root formation in Medicago truncatula.

Nodulation (Nod) factors (NFs) are symbiotic molecules produced by rhizobia that are essential for establishment of the rhizobium-legume endosymbiosis. Purified NFs can stimulate lateral root formation (LRF) in Medicago truncatula, but little is known about the molecular mechanisms involved. Using a combination of reporter constructs, pharmacological and genetic approaches, we show that NFs act on early steps of LRF in M. truncatula, independently of the ethylene signaling pathway and of the cytokinin receptor MtCRE1, but in interaction with auxin. We conducted a whole-genome transcriptomic study upon NF and/or auxin treatments, using a lateral root inducible system adapted for M. truncatula. This revealed a large overlap between NF and auxin signaling and, more interestingly, synergistic interactions between these molecules. Three groups showing interaction effects were defined: group 1 contained more than 1500 genes responding specifically to the combinatorial treatment of NFs and auxin; group 2 comprised auxin-regulated genes whose expression was enhanced or antagonized by NFs; and in group 3 the expression of NF regulated genes was antagonized by auxin. Groups 1 and 2 were enriched in signaling and metabolic functions, which highlights important crosstalk between NF and auxin signaling for both developmental and symbiotic processes.

Abscisic acid promotes pre-emergence stages of lateral root development in Medicago truncatula.

The plant root system is important for plant anchorage and nutrition. Among the different characteristics of the root system, root branching is a major factor of plasticity and adaptation to changing environments. Indeed, many biotic and abiotic stresses, such as drought or symbiotic interactions, influence root branching. Many studies concerning root development and root branching were performed on the model plant Arabidopsis thaliana, but this model plant has a very simplified root structure and is not able to establish any symbiotic interactions. We have recently described 7 stages for lateral root development in the model legume Medicago truncatula and found significant differences in the tissular contribution of root cell layers to the formation of new lateral roots (LR). We have also described 2 transgenic lines expressing the DR5:GUS and DR5:VENUS-N7 reporter genes that are useful to follow LR formation at early developmental stages. Here, we describe the use of these transgenic lines to monitor LR developmental responses of M. truncatula to the phytohormone abscisic acid (ABA) which is a major actor of stress and symbiotic interactions. We show that ABA promotes the formation of new lateral root primordia and their development, mostly at the late, pre-emergence stage.

Lateral root formation and patterning in Medicago truncatula.

The plant root system is crucial for anchorage and nutrition, and has a major role in plant adaptation, as well as in interactions with soil micro-organisms. Despite the agronomical and ecological importance of legume plants, whose roots can interact symbiotically with soil bacteria called rhizobia that fix atmospheric dinitrogen, and the evidence that lateral root (LR) development programmes are intercepted and influenced by symbiotic organisms, very little is known concerning the cellular and molecular events governing LR development in legumes. To better understand the interconnections between LR formation and symbiotic processes triggered by rhizobia or symbiotic molecules such as lipo-chitooligosaccharides (LCOs), we first need a detailed description of LR development mechanisms in legumes. Using thin sections, we have described the cellular events leading to the formation of a new LR primordium (LRP) in Medicago truncatula, and divided them into seven stages prior to LR emergence. To monitor auxin accumulation we generated transgenic DR5:GUS and DR5:VENUS-N7 reporter lines of M. truncatula, and used them to analyze early stages of LR development. Interesting differences were observed for LR ontogeny compared to Arabidopsis thaliana. Notably, we observed endodermal and cortical contributions to LRP formation, and the associated DR5:GUS expression profile indicated that endodermal and cortical cell divisions were correlated with auxin accumulation. As described for A. thaliana, we observed a preferential zone for LR initiation at 4.45 mm from the root tip. Finally, we studied LR emergence and showed that a significant proportion of new LRP do not emerge straight away and could thus be an additional source of root plasticity. Our results shed new light on the patterning and early development of LRs in M. truncatula.