A Low-volume Hydroponic Protocol for Maize and A Sensitive Bud-length Assay for Root-to-Shoot Impacts of The Strigolactone Analog, rac-GR24

Background

The initial purpose for developing the protocol presented here was to quantify root-to-shoot impacts of the hormone, strigolactone (SL) in maize. A central role was postulated for strigolactone in domestication of this species, due to contributions by this hormone to plant architecture and thus the single-stalk morphology of modern-day maize (Guan et al., 2012). To address this possibility, a system was needed that would 1) allow root-feeding studies throughout initial vegetative growth of this large grass species, 2) minimize amount and cost of the SL analog to be used (rac-GR24), and 3) allow accurate quantification of shoot responses in terms of lateral-bud outgrowth. The resulting protocol can be adapted to a wide range of root-feeding, root-labeling, and exudate-sampling studies with low-volume hydroponic growth of large, C4 grass species. A new approach for strigolactone study in these species is also provided.

Strigolactones (SLs) contribute to apical dominance in diverse species (Wang et al., 2018). A similar suppression of lateral shoots is a key domestication feature in maize. The single-stalk architecture results from elevated expression of a domestication gene, Teosinte branched 1 (Tb1) (Doebley et al., 1997). Under normal conditions, lateral buds are tiny, protected by leaf sheaths, and remain dormant in most maize inbreds. The buds do not elongate into visible tillers unless apical dominance is released by decapitation or especially favorable growth conditions. The possible role of SLs in this process emerged with their identification as phytohormones involved in axillary branching (Gomez-Roldan et al., 2008; Umehara et al., 2008). Direct contributions in maize were revealed by the transposon insertion into a gene for SL biosynthesis (Zea mays Carotenoid Cleavage Dioxygenase 8 (ZmCCD8). The resulting, SL-deficient maize mutant, zmccd8, showed characteristics of a primitive, pre-domestication phenotype that included outgrowth of lateral buds and limited apical dominance (Guan et al., 2012).

Although genes for SL biosynthesis are expressed in many tissues, most of its formation occurs in roots. The root-derived SLs are transported acropetally to shoots through xylem (Kohlen et al., 2010) and via ABC transporters (Sasse et al., 2015). Plant roots also secrete SLs into the rhizosphere, where they attract symbiotic partners such as arbuscular mycorrhizal fungi (Akiyama et al., 2005) and nitrogen-fixing bacteria (López-Ráez et al., 2017). Unfortunately, the SL exudates are also sensed by seeds of parasitic weeds like Striga that use these secretions as signals to germinate and locate host roots (Aliche et al., 2020). The discovery of diverse SL functions enhances its potential applications in agriculture (Aliche et al., 2020).

Hydroponic systems have been used to study roles of SLs in axillary branching by Arabidopsis (Waters et al., 2012) and rice (Umehara et al., 2008). This approach has also been widely used to investigate homeostasis of mineral nutrients and processes that include iron uptake in maize (Nozoye et al., 2013). However, analysis of SL effects on lateral buds in maize seedlings required improvements in the efficiency of existing hydroponic protocols. Two paramount considerations were 1) the expense of rac-GR24, and 2) the quantity needed to support development of lateral buds. The synthetic SL analog, rac-G24, is costly as well as short-lived, with a half-life around 3 days in hydroponic solution. Supplies must thus be refreshed every 3 to 4 days, and the hydroponic system must have a low enough solution volume to minimize usage of rac-GR24. At the same time, the compact system must also provide enough space for growth of the lateral buds and roots. The bud outgrowth in particular, is sensitive to changes in nutrient availability and growing space. The protocol described here addresses the above requirements. It can also be used to advance other research in the field of root biology, plant-microbe interactions, crop-weed interactions, and mechanisms of mineral-nutrient homeostasis in grass crops.