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Stomata are the tiny gateways that regulate gas exchange between the atmosphere and the internal tissues of the plant. Serving both as entry points for CO2 and as exit points for water vapor, stomata have important roles in photosynthesis and transpiration. Survival of the plant depends on its ability to open or close stomata rapidly in response to environmental and endogenous stimuli. For instance, excessive water vapor loss would result in dehydration, and it is thus critical that stomata close under dry conditions. Stomatal closure is mediated by a complex molecular cascade that culminates in the reduction of turgor pressure in the pair of guard cells surrounding the stomatal pore. The actin cytoskeleton of guard cells is remodeled during stomatal closure, changing from radial filaments in open guard cells to dense longitudinal bands in closed ones (Eun and Lee, 1997), and has long since been known to function in stomatal aperture regulation (Kim et al., 1995). However, the mechanism by which the actin cytoskeleton is remodeled during stomatal closure and the purpose of this remodeling are unclear.
Zhao et al. (2016) recently identified a mutant line harboring an insertion in the gene encoding CASEIN1-LIKE PROTEIN2 (CKL2) in a screen for mutants that wilt more readily than the wild type and are less sensitive to abscisic acid (ABA)-induced stomatal closure. The authors found that CKL2, which is expressed in many tissues of the plant, is induced by both ABA and water-loss treatment. To examine the subcellular localization of CKL2, the authors transformed both wild-type and ckl2 mutant plants with a CKL2pro:GFP-CKL2 construct. The construct rescued the stomatal closure defect and drought-sensitive phenotype of the mutant. The finding that GFP-labeled CKL2 formed filamentous networks in several cell types, including guard cells, suggested that CKL2 associates with cytoskeletal elements. Pharmacological studies showed that CKL2 colocalized with actin filaments, but not with microtubules. In addition, GFP-labeled CKL2 colocalized with rhodamine-phalloidin-stained actin filaments in a cell suspension. However, a cosedimentation assay showed that CKL2 does not bind to actin in vitro.
A comparison of fluorescently labeled actin microfilaments in wild-type and ckl2 guard cells showed that the actin filaments of the mutant were disrupted more rapidly by treatment with the actin-destabilizing drug latrunculin A than were those of the wild type, suggesting that CKL2 stabilizes actin filaments in guard cells. Further analysis showed that this actin-stabilizing activity of CKL2 is essential for the construction of longitudinal actin cables during ABA-induced stomatal closure. Finally, the authors showed that CKL2 interacts with and phosphorylates actin depolymerizing factor 4 (ADF4), thereby blocking its actin filament disassembly activity.
Based on these findings, the authors propose that CKL2 regulates actin cytoskeleton remodeling during stomatal closure by phosphorylating ADF4 (see figure). Future research should examine how actin filament remodeling affects cellular processes in guard cells to bring about changes in stomatal aperture.
Simplified model of ABA-induced stomatal closure. Actin microfilaments undergo dramatic changes during stomatal closure, changing from radial filaments in open stomata (left), to a disorganized array in closing stomata (middle), to thick longitudinal cables in closed stomata (right). ABA-induced CKL2 blocks ADF activity late in this process, thereby stabilizing actin microfilaments. (Adapted from Zhao et al. [2016], Figure 8.)
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