ABA guides stomatal proliferation and patterning through the EPF-SPCH signaling pathway in Arabidopsis thaliana.

Adaptation to dehydration stress requires plants to coordinate environmental and endogenous signals to inhibit stomatal proliferation and modulate their patterning. The stress hormone abscisic acid (ABA) induces stomatal closure and restricts stomatal lineage to promote stress tolerance. Here, we report that mutants with reduced ABA levels, xer-1, xer-2 and aba2-2, developed stomatal clusters. Similarly, the ABA signaling mutant snrk2.2/2.3/2.6, which lacks core ABA signaling kinases, also displayed stomatal clusters. Exposure to ABA or inhibition of ABA catabolism rescued the increased stomatal density and spacing defects observed in xer and aba2-2, suggesting that basal ABA is required for correct stomatal density and spacing. xer-1 and aba2-2 displayed reduced expression of EPF1 and EPF2, and enhanced expression of SPCH and MUTE. Furthermore, ABA suppressed elevated SPCH and MUTE expression in epf2-1 and epf1-1, and partially rescued epf2-1 stomatal index and epf1-1 clustering defects. Genetic analysis demonstrated that XER acts upstream of the EPF2-SPCH pathway to suppress stomatal proliferation, and in parallel with EPF1 to ensure correct stomatal spacing. These results show that basal ABA and functional ABA signaling are required to fine-tune stomatal density and patterning.

Stomata in Close Contact: The Case of Pancratium maritimum L. (Amaryllidaceae).

A special feature found in Amaryllidaceae is that some guard cells of the neighboring stomata form a "connection strand" between their dorsal cell walls. In the present work, this strand was studied in terms of both its composition and its effect on the morphology and function of the stomata in Pancratium maritimum L. leaves. The structure of stomata and their connection strand were studied by light and transmission electron microscopy. FM 4-64 and aniline blue staining and application of tannic acid were performed to detect cell membranes, callose, and pectins, respectively. A plasmolysis experiment was also performed. The composition of the connection strand was analyzed by fluorescence microscopy after immunostaining with several cell-wall-related antibodies, while pectinase treatment was applied to confirm the presence of pectins in the connection strand. To examine the effect of this connection on stomatal function, several morphological characteristics (width, length, size, pore aperture, stomatal distance, and cell size of the intermediate pavement cell) were studied. It is suggested that the connecting strand consists of cell wall material laid through the middle of the intermediate pavement cell adjoining the two stomata. These cell wall strands are mainly comprised of pectins, and crystalline cellulose and extensins were also present. Connected stomata do not open like the single stomata do, indicating that the connection strand could also affect stomatal function. This trait is common to other Amaryllidaceae representatives.

Effects of stomata clustering on leaf gas exchange.

A general theoretical framework for quantifying the stomatal clustering effects on leaf gaseous diffusive conductance was developed and tested. The theory accounts for stomatal spacing and interactions among 'gaseous concentration shells'. The theory was tested using the unique measurements of Dow et al. (2014) that have shown lower leaf diffusive conductance for a genotype of Arabidopsis thaliana with clustered stomata relative to uniformly distributed stomata of similar size and density. The model accounts for gaseous diffusion: through stomatal pores; via concentration shells forming at pore apertures that vary with stomata spacing and are thus altered by clustering; and across the adjacent air boundary layer. Analytical approximations were derived and validated using a numerical model for 3D diffusion equation. Stomata clustering increases the interactions among concentration shells resulting in larger diffusive resistance that may reduce fluxes by 5-15%. A similar reduction in conductance was found for clusters formed by networks of veins. The study resolves ambiguities found in the literature concerning stomata end-corrections and stomatal shape, and provides a new stomata density threshold for diffusive interactions of overlapping vapor shells. The predicted reduction in gaseous exchange due to clustering, suggests that guard cell function is impaired, limiting stomatal aperture opening.