Quantitative detection of the maize phytocytokine Zip1 utilizing ELISA

Plant signaling peptides, also known as phytocytokines, play a crucial role in cell-to-cell communication during plant development and immunity. The detection of small peptides in plant tissues is challenging and often relies on time-consuming and cost-intensive approaches. Here, we present an ELISA-based assay as a rapid and cost-effective method for the detection of naturally released peptides in plant tissues. Our ELISA-based method was developed to detect Zip1, a 17-amino-acid phytocytokine derived from Zea mays that elicits salicylic acid signaling in maize leaves. Using a custom peptide-antibody, we designed an experimental pipeline to achieve peptide specificity, selectivity, and sensitivity allowing the detection of the Zip1 peptide in complex biological samples. As a proof of concept, we first overexpressed the precursor molecule PROZIP1 in Nicotiana benthamiana and in transfected maize protoplasts and monitored the release of Zip1-containing peptides. In a second approach we treated maize leaves with salicylic acid to induce native PROZIP1 expression and processing. Using ELISA, we were able to quantify native Zip1 signals with a detection limit in the nanogram range, which allowed us to detect different Zip1-containing peptides in plant material. This method can be adapted for the detection and quantification of a variety of plant signaling peptides.