Koenig Maurice, Sorger Zarah, Keh Shania Pin Yin, Doehlemann Gunther, Misas Villamil Johana C
Institute for Plant Sciences, University of Cologne, Cologne, Germany.
Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne, Cologne, Germany.
J Exp Bot. 2025 Jan 10;76(2):299-311. doi: 10.1093/jxb/erae423.
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.
植物信号肽，也被称为植物细胞分裂素，在植物发育和免疫过程中的细胞间通讯中起着至关重要的作用。在植物组织中检测小肽具有挑战性，通常依赖于耗时且成本高昂的方法。在此，我们提出一种基于酶联免疫吸附测定（ELISA）的检测方法，作为一种快速且经济高效的检测植物组织中天然释放肽的方法。我们基于ELISA的方法是为检测Zip1而开发的，Zip1是一种来自玉米的由17个氨基酸组成的植物细胞分裂素，可在玉米叶片中引发水杨酸信号传导。我们使用定制的肽抗体设计了一个实验流程，以实现肽的特异性、选择性和敏感性，从而能够在复杂的生物样品中检测Zip1肽。作为概念验证，我们首先在本氏烟草和转染的玉米原生质体中过表达前体分子PROZIP1，并监测含Zip1肽的释放情况。在第二种方法中，我们用水杨酸处理玉米叶片以诱导天然PROZIP1的表达和加工。通过ELISA，我们能够以纳克范围内的检测限对天然Zip1信号进行定量，这使我们能够检测植物材料中不同的含Zip1肽。该方法可适用于多种植物信号肽的检测和定量。
