Analysis of Guard Cell Readouts Using Arabidopsis thaliana Isolated Epidermal Peels.

Stomata are pores surrounded by a pair of specialized cells, called guard cells, that play a central role in plant physiology through the regulation of gas exchange between plants and the environment. Guard cells have features like cell-autonomous responses and easily measurable readouts that have turned them into a model system to study signal transduction mechanisms in plants. Here, we provide a detailed protocol to analyze different physiological responses specifically in guard cells. We describe, in detail, the steps and conditions to isolate epidermal peels with tweezers and to analyze i) stomatal aperture in response to different stimuli, ii) cytosolic parameters such as hydrogen peroxide (H2O2), glutathione redox potential (E GSH), and MgATP-2 in vivo dynamics using fluorescent biosensors, and iii) gene expression in guard cell-enriched samples. The importance of this protocol lies in the fact that most living cells on epidermal peels are guard cells, enabling the preparation of guard cell-enriched samples. Key features • Isolation of epidermal peels as a monolayer enriched in guard cells • Measurement of cytosolic guard cell signaling component dynamics in isolated epidermal peels through fluorescent biosensor analysis • Gene expression analysis of guard cell-enriched isolated tissue.

Mitochondrial H2S donor AP39 induces stomatal closure by modulating guard cell mitochondrial activity.

Hydrogen sulfide (H2S) is a gaseous signaling molecule involved in numerous physiological processes in plants, including gas exchange with the environment through the regulation of stomatal pore width. Guard cells (GCs) are pairs of specialized epidermal cells that delimit stomatal pores and have a higher mitochondrial density and metabolic activity than their neighboring cells. However, there is no clear evidence on the role of mitochondrial activity in stomatal closure induction. In this work, we showed that the mitochondrial-targeted H2S donor AP39 induces stomatal closure in a dose-dependent manner. Experiments using inhibitors of the mitochondrial electron transport chain (mETC) or insertional mutants in cytochrome c (CYTc) indicated that the activity of mitochondrial CYTc and/or complex IV are required for AP39-dependent stomatal closure. By using fluorescent probes and genetically encoded biosensors we reported that AP39 hyperpolarized the mitochondrial inner potential (Δψm) and increased cytosolic ATP, cytosolic hydrogen peroxide levels, and oxidation of the glutathione pool in GCs. These findings showed that mitochondrial-targeted H2S donors induce stomatal closure, modulate guard cell mETC activity, the cytosolic energetic and oxidative status, pointing to an interplay between mitochondrial H2S, mitochondrial activity, and stomatal closure.

Hydrogen sulphide as a guard cell network regulator.

Hydrogen sulphide (H2 S) is an endogenously produced gasotransmitter that has rapidly emerged as an active signalling component of several plant processes, stomatal movement regulation among them. The guard cells (GCs), pairs of cells that neighbour the stomatal pores, transduce endogenous and environmental signals, through signalling network, to control stomatal pore size. In this complex network, which has become a model system for plant signalling, few highly connected components form a core that links most of the pathways. The evidence summarized in this insight, on the interplay between H2 S and different key components of the GC networks, points towards H2 S as a regulator of the GC core signalling pathway.