May the dark be with roots: Effects of root illumination in vitro experiments

The root system generates an underground network that anchors the plants to the soil, providies nutrients and water while creating a defence network and facilitating beneficial interactions with a multitude of living organisms and climatological conditions. In nature, roots grow into the soil, in a dark environment. However, to facilitate morphological and molecular studies, the majority of the root research has been conducted using in vitro systems. This illumination modifies the root biology, masking the natural root responses to environmental changes. In this viewpoint, we discuss the results found over the last decade on how experimental exposure of roots to light may bias root development and responses through the alteration of hormonal signalling, cytoskeleton organisation, reactive oxygen species (production or scavenging), or the accumulation of flavonoids, among other factors. Illumination alters the uptake of nutrients or water, and also affects the response of the roots to abiotic stresses. It should be highlighted the effect of root interactions with the soil microbiota. Furthermore, we review in vitro systems that have been designed to cultivate plants in vitro with roots in darkness Furthermore, a comparative analysis of root transcriptomes obtained with different devices that keep roots in darkness is provided. In addition, we also identified other experimental variables that should be considered to better mimic soil conditions, whose improvement would benefit studies using in vitro cultivation or enclosed ecosystems. In conclusion, to obtain close to natural conditions data, we believe, and recommend, that experiments should be done cultivating plants with the root system in darkness.