The use of quantitative imaging to investigate regulators of membrane trafficking in Arabidopsis stomatal closure

Abstract

Expansion of gene families facilitates robustness and evolvability of biological processes but impedes functional genetic dissection of signalling pathways. To address this, quantitative analysis of single cell responses can help characterize the redundancy within gene families. We developed high‐throughput quantitative imaging of stomatal closure, a response of plant guard cells, and performed a reverse genetic screen in a group of Arabidopsis mutants to five stimuli. Focussing on the intersection between guard cell signalling and the endomembrane system, we identified eight clusters based on the mutant stomatal responses. Mutants generally affected in stomatal closure were mostly in genes encoding SNARE and SCAMP membrane regulators. By contrast, mutants in RAB5 GTPase genes played specific roles in stomatal closure to microbial but not drought stress. Together with timed quantitative imaging of endosomes revealing sequential patterns in FLS2 trafficking, our imaging pipeline can resolve non‐redundant functions of the RAB5 GTPase gene family. Finally, we provide a valuable image‐based tool to dissect guard cell responses and outline a genetic framework of stomatal closure.

At the intersection of exocytosis and endocytosis in plants

“…Moreover, the aberrant stomatal function in the chc mutant is similar to the phenotype of the secretory SNARE SYP121 mutation that also impairs endocytosis, suggesting that coordination of endo‐ and exocytosis regulated by clathrin machinery and SNARE proteins is important for stomatal function (Larson et al ., ). The development of quantitative imaging for stomatal closure facilitates revealing the functions of trafficking regulators in stomatal movement (Bourdais et al ., ). Thus, plant physiological responses to certain abiotic stimuli involve a tradeoff between exo‐ and endocytic membrane trafficking to maintain PM integrity and stress tolerance.…”

Distinct EH domains of the endocytic TPLATE complex confer lipid and protein binding

“…SCAMP proteins were first characterized in mammals for their role in endocytosis (Fernández-Chacón et al, 2000). Current insights in the animal and plant field show a broader function of SCAMPs in plasma membrane phase separation, cell plate formation, and pathogen-induced stomatal closure (Bourdais et al, 2019;Lam et al, 2008;Park et al, 2018). SCAMP5 was also previously identified to reside in close proximity to TPC by proximity labelling (Arora et al, 2020).…”

Completing Autophagy: Formation and Degradation of the Autophagic Body and Metabolite Salvage in Plants

“…Plant VTI12 is the ortholog of yeast Vti1 (Figure 2, Table 1). In Arabidopsis mutants with T-DNA VTI12 insertion, growing in rich-nutrient conditions, presented a normal phenotype, whereas under nutrient-poor conditions accelerated aging was observed, confirming that VTI12 is involved in autophagy in plants [25,[74][75][76][77]. This protein also participates in the transport of storage proteins from the cytoplasm to the vacuole [76].…”