Size matters: three methods for estimating nuclear size in mycorrhizal roots of Medicago truncatula by image analysis

Gennaro Carotenuto, Ivan Sciascia, Ludovica Oddi, Veronica Volpe, Andrea Genre

The intracellular accommodation of arbuscular mycorrhizal (AM) fungi involves a profound molecular reprogramming of the host cell architecture and metabolism, based on the activation of a symbiotic signaling pathway. In analogy with other plant biotrophs, AM fungi are reported to trigger cell cycle reactivation in their host tissues, possibly in support of the enhanced metabolic demand required for the symbiosis. We here compare the efficiency of three Fiji/ImageJ image analysis plugins in localizing and quantifying the increase in nuclear size - a hallmark of recursive events of endoreduplication - in M. truncatula roots colonized by the AM fungus Gigaspora margarita. All three approaches proved to be versatile and upgradeable, allowing the investigation of nuclear changes in a complex tissue; 3D Object Counter provided more detailed information than both TrackMate and Round Surface Detector plugins. On this base we challenged 3D Object Counter with two case studies: verifying the lack of endoreduplication-triggering responses in Medicago truncatula mutants with a known non-symbiotic phenotype; and analysing the correlation in space and time between the induction of cortical cell division and endoreduplication upon AM colonization. Both case studies revealed important biological aspects. Mutant phenotype analyses have demonstrated that the knock-out mutation of different key genes in the symbiotic signaling pathway block AM-associated endoreduplication. Furthermore, our data show that cell divisions occur during initial stages of root colonization and are followed by recursive activation of the endocycle in preparation for arbuscule accommodation. In conclusion, our results indicate 3D Object Counter as the best performing Fiji/ImageJ image analysis script in plant root thick sections and its application highlighted endoreduplication as a major feature of the AM pre-penetration response in root cortical cells.