Heterologous expression of ZmWBC33 in Arabidopsis and protein localization with green fluorescent protein (GFP)

Strigolactones (SLs) are a group of molecules derived from carotenoids, recently classified as phytohormones, which were demonstrated to be involved in multiple endogenous processes of plant development and once exuded from the roots to the soil, in the regulation of interactions between the plant and the rhizosphere, particularly promoting symbiotic relationships with arbuscular mycorrhizal fungi. To date, more than 30 derivatives of SLs have been described as potentially interesting in basic science for the study of plant-rhizosphere interactions and in applied science for their use in agriculture to address the challenges of crop productivity and resilience and to promote sustainable agronomic practices. Much has been discovered about the biosynthesis and signalling processes of SLs, even showing that they are widely conserved among different plant species. However, little is known about their distribution within plants and their transport from root to soil, as well as their regulation and synchronisation with SL biosynthesis and nutrient availability in the soil. So far, the only transporter characterised is the petunia PDR1 protein, an ATP BINDING CASSETTE (ABC) protein and its putative orthologue PDR6 in Nicotiana tabacum. ZmWBC33 is a member of the WBC subfamily of maize ABCG transporters putatively encoding for a SL transporter in maize (Zea mays L.). Heterologous expression of the ZmWBC33 gene in Arabidopsis, led to the identification of 11 distinct transgenic lines, comprising those with and without the integration of the green fluorescent protein (GFP), that were and will be used to investigate the cellular WBC33 protein localization through confocal microscope, followed by the identification of potential new homozygous sublines to perform future physiological studies. The fluorescent signal was detected in the plasma membrane of all the tissues sampled, including the external layer of cortex cells in the root apex, the region above it, the outer part of lateral root primordium, the cortex cells of the root maturation zone surrounding the stele, and the plasma membrane of epidermis. These results confirm the achievement of the transformation process and further support the hypothesis that ZmWBC33 may be a SLs transporter. In parallel, a hydroponic growth system for Arabidopsis seedlings was developed in order to collect root exudates to perform a Phelipanche ramosa germination bioassay. By controlling nutrient availability and manipulating the concentrations of the elements of interest, it was possible to set up experiments to test the effects of nitrogen (N) and phosphorus (P) deficiency on the SLs content from 5-week-old seedlings. Results suggested that the use of a reduced volume (15 ml) to collect exudates is more suitable to obtain higher seed germination rates, and that under conditions of nutritional deficiencies of N and P plants tend to exude more SLs in the first 3 days of stress, compared to a prolonged period of 7 days.