Background

Starch is a complex polymer of glucose and represents the most abundant form in which plants store carbohydrate. Starch serves different functions, according to the cell types from which it is derived, and the external environmental conditions. In guard cells, which border the stomatal pores that control water and carbon dioxide exchange with the environment, starch can be mobilized within minutes upon transition to light, helping to generate organic acids and sugars to increase guard cell turgor and promote stomatal opening. In mesophyll cells, starch typically accumulates gradually during the day and is degraded at night to support metabolism (Santelia and Lunn, 2017).

Because guard cells comprise only a minor fraction of the total leaf, it is difficult to measure starch quantitatively using conventional methods. Up until now, starch accumulation in guard cells has been mostly visualized by iodine staining. This technique can determine the presence/absence of starch but does not provide accurate, quantitative information.

Here, we describe a fluorescence-based imaging method to quantify starch in guard cells of Arabidopsis thaliana. This technique is based on the covalent labeling of cell wall material and other glucan substrates, including starch, with the fluorescent pseudo-Schiff reagent propidium iodide (PS-PI). Isolated epidermal peels are treated with periodic acid to oxidize the hydroxyl groups of the glucose units to aldehyde and ketone groups. The aldehyde groups (-CHO) can then react covalently with propidium iodide, resulting in samples with highly fluorescent glucans that are well suited for confocal laser scanning microscopy. The area of single starch granules within guard cell chloroplasts can be determined using digital imaging. We applied this method to assess the dynamics of starch content in guard cells of intact Arabidopsis leaves over the diurnal cycle, and to determine the impact of fusicoccin (a chemical activator of the proton pump) on starch amounts in guard cells of isolated epidermal peels fragments floating in stomatal opening buffer (Horrer et al., 2016).

Our protocol is an adaptation of a previous mPS-PI staining technique (Truernit et al., 2008). The original method was developed to image entire plant organs for three-dimensional reconstruction of their cellular organization. The main difference between the two methods is the incubation time with the propidium iodide solution, which is 1-2 h for entire plant organs, and only about 20-40 min for epidermal peels.

The technique described here is simple, accurate and highly reproducible. By facilitating the detailed quantification of starch amounts in guard cells, this method will increase the number of questions we will be able to answer about any aspect of guard cell starch metabolism.