Split-root, grafting and girdling as experimental tools to study root-to shoot-to root signaling

Abstract Plant growth is related to the amount of nutrients that it can acquire and it can be limited by poor root development and nutrient uptake, and low efficiency of nutrient use. Nutrients are not distributed evenly throughout the soil around roots, causing plants to put in action the functioning of the various local and systemic signaling pathways to coordinate adaptative responses to the most diverse types of stresses, among them nutritional limitation. A coordinated array of long-distance signaling mechanisms integrates the communication between shoots and roots, which provide photoassimilates and nutrients, respectively. The use of techniques to study signaling among the most diverse plant species has gained prominence. The main techniques for studying nutrient signaling between shoots and roots are split-root, grafting and girdling. The split-root system is characterized by separating a single root system in two independent compartments, with a common shoot. Split-root has been used for analyzing transcriptional, biochemical, and physiological changes in roots in response to nutritional challenges. Grafting has stood out as an important method concerning signaling mechanisms studies. It still follows the basic principle of cutting and fitting two parts (scion = bud and rootstock = root) from different plants, which merge, grow and develop as a single plant. Most recent studies have been in detecting long-distance signals, their transport, and the factors involved in signaling mechanisms, with particular attention to the role of phytohormones, RNAs, peptides, and proteins. Girdling is an important technique widely used in fruit trees. It consists of the removal of a strip of bark from branches or trunk, affecting different stages of reproductive growth, including flowering and fruiting, development, ripening, and nutritional quality of fruits. The increase in the amount of carbohydrates above the cut induces the expression of genes involved in the metabolism of carbohydrates, favoring the respiratory potential of the stem and branches, as well as the leaf mass per area. The techniques reviewed here allow the monitoring and study of long-distance signaling and relate it to plant development through simple, efficient, and reproducible approaches. They permit researchers to make inferences about the existence, direction, and intensity of the cross-talk between long-distance signaling and nutrients, suggesting that mobile signals and their interactions with individual nutrients exist.