Kamleitner Marília, Strauß Annett, Faiß Natalie, Lami Klea, Lahaye Thomas, Schaaf Gabriel, Gaugler Verena
Department of Plant Nutrition, Institute of Crop Science and Resource Conservation (INRES), University of Bonn, Bonn, Germany.
Department of General Genetics, Center of Plant Molecular Biology (ZMBP) University of Tübingen, Tübingen, Germany.
Methods Mol Biol. 2025;2972:235-247. doi: 10.1007/978-1-0716-4799-8_17.
In recent years, there has been substantial progress in the development of methods to analyze inositol phosphates (InsPs) and inositol pyrophosphates (PP-InsPs). However, many of these techniques are labor- and cost-intensive and can usually only be carried out by laboratories specialized in InsPs/PP-InsPs analysis. In this chapter, we present a simple method that exploits the fact that phosphorylation and/or dephosphorylation of certain InsP/PP-InsP species induces the activation of promoters driving the expression of genes involved in phosphate starvation response (PSR). By linking PSR-inducible promoters to the RUBY reporter, which allows continuous and noninvasive visualization of promoter activation, we provide a new analytical tool to monitor enzymatic activities that alter PP-InsP levels, eliminating the need for complex biochemical analysis. We present a step-by-step protocol showing how simple coinfiltration of promoter-RUBY T-DNA constructs together with PP-InsP pyrophosphatase-encoding T-DNAs into Nicotiana benthamiana leaves enables evaluation of the enzymatic activity of expressed pyrophosphatases, and mention possible downstream analyses by methods described in other chapters of this special issue.
近年来，在分析肌醇磷酸（InsPs）和肌醇焦磷酸（PP-InsPs）的方法开发方面取得了重大进展。然而，这些技术中的许多都需要耗费大量人力和成本，并且通常只能由专门从事InsPs/PP-InsPs分析的实验室进行。在本章中，我们介绍了一种简单的方法，该方法利用了某些InsP/PP-InsP物种的磷酸化和/或去磷酸化会诱导驱动参与磷饥饿反应（PSR）的基因表达的启动子激活这一事实。通过将PSR诱导型启动子与红宝石报告基因相连，从而能够对启动子激活进行连续且非侵入性的可视化监测，我们提供了一种新的分析工具来监测改变PP-InsP水平的酶活性，无需复杂的生化分析。我们展示了一个逐步方案，说明如何将启动子-红宝石T-DNA构建体与编码PP-InsP焦磷酸酶的T-DNA简单地共浸润到本氏烟草叶片中，从而评估所表达焦磷酸酶的酶活性，并提及了本期特刊其他章节中所述方法可能进行的下游分析。
