HSP70-3 Interacts with Phospholipase Dδ and Participates in Heat Stress Defense.

Heat shock proteins (HSPs) function as molecular chaperones and are key components responsible for protein folding, assembly, translocation, and degradation under stress conditions. However, little is known about how HSPs stabilize proteins and membranes in response to different hormonal or environmental cues in plants. Here, we combined molecular, biochemical, and genetic approaches to elucidate the involvement of cytosolic HSP70-3 in plant stress responses and the interplay between HSP70-3 and plasma membrane (PM)-localized phospholipase Dδ (PLDδ) in Arabidopsis (Arabidopsis thaliana). Analysis using pull-down, coimmunoprecipitation, and bimolecular fluorescence complementation revealed that HSP70-3 specifically interacted with PLDδ. HSP70-3 bound to microtubules, such that it stabilized cortical microtubules upon heat stress. We also showed that heat shock induced recruitment of HSP70-3 to the PM, where HSP70-3 inhibited PLDδ activity to mediate microtubule reorganization, phospholipid metabolism, and plant thermotolerance, and this process depended on the HSP70-3-PLDδ interaction. Our results suggest a model whereby the interplay between HSP70-3 and PLDδ facilitates the re-establishment of cellular homeostasis during plant responses to external stresses and reveal a regulatory mechanism in regulating membrane lipid metabolism.

Involvement of Arabidopsis phospholipase D δ in regulation of ROS-mediated microtubule organization and stomatal movement upon heat shock.

Reactive oxygen species (ROS) are plant metabolic and signaling molecules involved in responses to various external stresses, but the existence of ROS receptors and how plants respond to ROS remain largely unknown. Here we report that the plasma membrane-localized phospholipase D δ (PLDδ) protein is crucial for sensing heat shock-induced ROS to initiate reorganization of guard cell microtubules in Arabidopsis cotyledons. Heat shock of wild-type Arabidopsis cotyledons stimulated ROS production which disrupted microtubule organization and induced stomatal closure, whereas this process was markedly impaired in pldδ mutants. Moreover, wild-type PLDδ, but not the Arg622-mutated PLDδ, complemented the pldδ phenotypes in heat shock-treated plants. ROS activated PLDδ by oxidizing cysteine residues, an action that was required for its functions in ROS-induced depolymerization of guard cell microtubules, stomatal closure, and plant thermotolerance. Additionally, lipid profiling reveals involvement of microtubule organization in the feedback regulation of glycerolipid metabolism upon heat stress. Together, our findings highlight a potential mechanosensory role for PLDδ in regulating the dynamic organization of microtubules and stomatal movement, as part of the ROS-sensing pathway, during the response to external stresses.

Dynamic responses of PA to environmental stimuli imaged by a genetically encoded mobilizable fluorescent sensor.

Membrane fluidity, permeability, and surface charges are controlled by phospholipid metabolism and transport. Despite the importance of phosphatidic acid (PA) as a bioactive molecule, the mechanical properties of PA translocation and subcellular accumulation are unknown. Here, we used a mobilizable, highly responsive genetically encoded fluorescent indicator, green fluorescent protein (GFP)-N160RbohD, to monitor PA dynamics in living cells. The majority of GFP-N160RbohD accumulated at the plasma membrane and sensitively responded to changes in PA levels. Cellular, pharmacological, and genetic analyses illustrated that both salinity and abscisic acid rapidly enhanced GFP-N160RbohD fluorescence at the plasma membrane, which mainly depended on hydrolysis of phospholipase D. By contrast, heat stress induced nuclear translocation of PA indicated by GFP-N160RbohD through a process that required diacylglycerol kinase activity, as well as secretory and endocytic trafficking. Strikingly, we showed that gravity triggers asymmetric PA distribution at the root apex, a response that is suppressed by PLDζ2 knockout. The broad utility of the PA sensor will expand our mechanistic understanding of numerous lipid-associated physiological and cell biological processes and facilitate screening for protein candidates that affect the synthesis, transport, and metabolism of PA.