Split-root systems: detailed methodology, alternative applications, and implications at leaf proteome level

Abstract

Background Split-root systems (SRS) have many applications in plant sciences, but their implementation, depending on the experimental design, can be difficult and time-consuming. Additionally, the system is not exempt from limitations, since the time required for the establishment of the SRS imposes a limit to how early in plant development experiments can be performed. Here, we optimized and explained in detail a method for establishing a SRS in young Arabidopsis thaliana seedlings, both in vitro and in soil. Results We found that the partial de-rooting minimized the recovery time compared to total de-rooting, thus allowing the establishment of the split-root system in younger plants. Analysis of changes in the Arabidopsis leaf proteome following the de-rooting procedure highlighted the distinct metabolic alterations that totally and partially de-rooted plants undergo during the healing process. This system was also validated for its use in drought experiments, as it offers a way to apply water-soluble compounds to plants subjected to drought stress. By growing plants in a split-root system with both halves being water-deprived, it is possible to apply the required compound to one half of the root system, which can be cut from the main plant once the compound has been absorbed, thus minimizing rehydration and maintaining drought conditions. Conclusions Partial de-rooting is the suggested method for obtaining split-root systems in small plants like Arabidopsis thaliana, as growth parameters, survival rate, and proteomic analysis suggest that is a less stressful procedure than total de-rooting, leading to a final rosette area much closer to that of uncut plants. Additionally, we provide evidence that split root-systems can be used in drought experiments where water-soluble compounds are applied with minimal effects of rehydration.

Asymmetrical copper root pruning may improve root traits for reforesting steep and/or windy sites

“…Furthermore, no differences between treated and untreated portions were measured for the mean diameter of the root population clearly indicating that the root lengthening was responsible for the increment of the volume and biomass. This differentiated response of the root system has already been observed in split-root system experiments, which provides a way to simulate the heterogeneity inherent to field conditions (Fernández et al 2021). Finally, also in this case, our results indicate both a local and systemic response of the root system to the local Cu application.…”

CORONATINE INSENSITIVE 1-mediated repression of immunity-related genes in Arabidopsis roots is overcome upon infection withVerticillium longisporum

“…Systemic signalling in roots involves the synthesis of signal(s) travelling from the root to the shoot and back to the root (Ohkubo et al ., 2017; Wang et al ., 2019). In order to challenge the hypothesis of ‘systemic lifting of the repressive COI1 activity’, we performed experiments with split roots where the two halves of the root system were cultivated in different pots (Saiz-Fernandez et al ., 2021) (Supplementary Fig. S3).…”

Competition for light induces metal accumulation in a metal hyperaccumulating plant

“…In May 2016, newly grown ramets of A. halleri were selected and severed from eight mother plants per population and each ramet was grown in water-filled containers in the greenhouse to induce root formation. After two weeks, the ramets produced 6-10 roots, out of which all except two similarly-sized roots were severed (Kassaw and Frugoli 2012;Saiz-Fernández et al 2021). Each ramet was then grown in a split-root setup of paired 0.05 L pots with one root in a high-metal pot (with soil from metalliferous sites) and the other in a low-metal pot (with soil from non-metalliferous sites) (Fig.…”