The transthylakoid pH gradient (delta pH) plays several critical roles in photosynthesis. It drives the synthesis of ATP, controls photosynthetic electron transfer by slowing key reactions, and triggers feedback regulatory systems that protect reaction centers against photodamage. In addition, large delta pH values are thought to sensitize photosystem II reaction centers to photoinhibition. Despite a large number of studies, the quantitative relationships between delta pH and the processes it affects are poorly defined. The proposed work aims to address these issues by developing non-invasive probes of lumen and stromal pH to both in vitro and in vivo systems. Preliminary work from other laboratories, as well as ours, has demonstrated that mutant versions of the Aequorea victoria green fluorescent protein (GFP) can be used as rapidly responding intracellular pH probes, with a useful pH-sensitive ranges. Expression of these probes will be targeted to the stromal and lumenal compartments of tobacco plants, allowing us to non-invasively probe the pH of these compartments. We will then conduct experiments, using a prototype fluorimeter, to assess the utility of these probes to the investigation of pH changes in the lumen and stroma.
