The main activity consisted in morphological, physiological and transcriptomics analysis of the root response on the VHN system. The VHN system allows us to detect systemic “N-demand” effect on lateral root (LR) length in both top (R1) and bottom (R2) parts of the root system in the wild-type (Fig.3A). We also showed that the primary root (PR) length responds to “N-demand”. These results indicate that the VHN system is appropriate to study the root foraging response to heterogeneous distribution of N in the growth medium. In a nitrate sensor KO mutant, LR “N-demand” response is affected in the top (R1) part of the root (Fig.3B). This indicates that this sensor is part of the systemic response to heterogeneous N-environment. After only 8h on VHN, shoots accumulate gradually increasing quantities of N depending on the conditions (Ctr+N > N-top > N-tip > Ctr-N) (Fig.4A). The bottom part of the root (R2) is more efficient in N-accumulation than the top (R1) but the later has a higher capacity (Fig.4A to 4C). Nevertheless, after 4 days, plant biomass is similar in Ctr+N and heterogeneous N-conditions (N-top, N-tip) (Fig.4E). These data indicate that plants submitted to heterogeneous N-supply adjust their internal N-status to maintain growth at a level comparable to that of plants grown on homogeneous N-supply. Differential gene expression analysis proved that a systemic N-response is detectable as soon as 2h in both shoots and roots (Fig.5 and 8). However, this systemic response is larger at 8h (Fig.5 and 8) and is largely consistent with response to N in general and heterogeneous N in particular. In roots, we found different dynamics in gene responses to heterogeneous N depending on where N is supplied along the main root axis; systemic N-response is faster in R2 than R1. Moreover, at each time point, the DEG are overall mostly specific to each part of the root but gene expression patterns and biological functions are very similar.
The analysis of the gene expression patterns at 8h in the different tissues converge to indicate that various systemic signals co-exist. We identified 3 root-to-shoot signals (Fig.9 and 11): a N-supply signal, a N-demand signal and a “N at tip” signal coming from the root apex and indicating to the shoot whether N is present of not (Fig. 10). The latter signals is independent of any nutritional consideration and represent a so far unknown and very specific systemic signaling pathway. We showed that “N at tip signal” is dependent on root apex (3mm) and on the nitrate sensor previously mentioned. Besides, we discovered that shoots seem to “sense” how much N has been accumulated in aerial parts and to regulate genes accordingly (Fig. 9 and 11 “N-dose” pattern). In addition, shoots would integrate the “N-status” information with the 3 ascending signals and would send an integrative signal back to the roots (N-repletion, N-depletion, heterogeneous N, etc.) (Fig.7). In roots, those systemic signals would be integrated with local one to adjust gene expression.
The dissemination of these results will be done through the publication of a scientific article in one of the best journal. This article is currently under writing. After the publication, the question of a root tip signal directly transduced to the shoots will be further explored. Notably, we will address the respective role of the root and shoot apical meristems by exploiting the shoot transcriptional responses highlighted in this project.
