Quantitative detection of plant signaling peptides utilizing ELISA

Plant signaling peptides, also known as phytocytokines, are involved in a number of signaling mechanisms, including 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 transfected maize protoplasts to overexpress the precursor molecule PROZIP1 and treated maize leaves with salicylic acid to induce native PROZIP1 expression and Zip1 release. 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.