4 research outputs found
In vivo light sheet fluorescence microscopy of calcium oscillations in arabidopsis thaliana
No full textCalcium imaging in plants requires a high-resolution microscope, able to perform volumetric acquisition in a few seconds, inducing as low photobleaching and phototoxicity as possible to the sample. Light sheet fluorescence microscopy offers these capabilities, with the further chance to mount the sample in vertical position, mimicking the plant’s growth and physiological conditions. A protocol for plant preparation and mounting in a light sheet microscope is presented. First, the growth of Arabidopsis thaliana in a sample holder compatible with light sheet microscopy is described. Then, the requirements for sample alignment and image acquisition are detailed. Finally, the image processing steps to analyze calcium oscillations are discussed, with particular emphasis on ratiometric calcium imaging in Arabidopsis root hairs
Endoplasmic reticulum-localized CCX2 is required for osmotolerance by regulating ER and cytosolic Ca2+ dynamics in Arabidopsis
No full textCa2+ signals in plant cells are important for adaptive responsesto environmental stresses. Here, we report that the ArabidopsisCATION/Ca2+ EXCHANGER2 (CCX2), encoding a putative novelcation/Ca2+ exchanger that localizes to the endoplasmic reticulum,is strongly induced by salt and osmotic stresses. Compared withthe wild type, CCX2 loss- and gain-of-function mutants were lessand more tolerant to different osmotic stress, respectively, anddisplayed the most noteworthy phenotypes during salt stress.Remarkably, Cameleon Ca2+ sensors revealed that CCX2 activityimpacts cytosolic and Endoplasmic Reticulum (ER) Ca2+ concentrations.To our knowledge, ccx2 is the first plant mutant with ameasured alteration in ER Ca2+ concentrations. In this study, weidentified CCX2 as a pivotal player in the regulation of ER Ca2+dynamics that heavily impinge on plant growth upon salt stress.SCOPUS: ar.jinfo:eu-repo/semantics/publishe
The structural bases for agonist diversity in an Arabidopsis thaliana glutamate receptor-like channel
No full textArabidopsis thaliana glutamate receptor-like (GLR) channels are amino acid-gated ion channels involved in physiological processes including wound signaling, stomatal regulation, and pollen tube growth. Here, fluorescence microscopy and genetics were used to confirm the central role of GLR3.3 in the amino acid-elicited cytosolic Ca2+ increase in Arabidopsis seedling roots. To elucidate the binding properties of the receptor, we biochemically reconstituted the GLR3.3 ligand-binding domain (LBD) and analyzed its selectivity profile; our binding experiments revealed the LBD preference for L-Glu but also for sulfur-containing amino acids. Furthermore, we solved the crystal structures of the GLR3.3 LBD in complex with 4 different amino acid ligands, providing a rationale for how the LBD binding site evolved to accommodate diverse amino acids, thus laying the grounds for rational mutagenesis. Last, we inspected the structures of LBDs from nonplant species and generated homology models for other GLR isoforms. Our results establish that GLR3.3 is a receptor endowed with a unique amino acid ligand profile and provide a structural framework for engineering this and other GLR isoforms to investigate their physiology
The EF-hand Ca2+ binding protein MICU choreographs mitochondrial Ca2+ dynamics in Arabidopsis
No full textPlant organelle function must constantly adjust to environmental conditions, which requires dynamic coordination. Ca2+ signaling may play a central role in this process. Free Ca2+ dynamics are tightly regulated and differ markedly between the cytosol, plastid stroma, and mitochondrial matrix. The mechanistic basis of compartment-specific Ca2+ dynamics is poorly understood. Here, we studied the function of At-MICU, an EF-hand protein of Arabidopsis thaliana with homology to constituents of the mitochondrial Ca2+ uniporter machinery in mammals. MICU binds Ca2+ and localizes to the mitochondria in Arabidopsis. In vivo imaging of roots expressing a genetically encoded Ca2+ sensor in the mitochondrial matrix revealed that lack of MICU increased resting concentrations of free Ca2+ in the matrix. Furthermore, Ca2+ elevations triggered by auxin and extracellular ATP occurred more rapidly and reached higher maximal concentrations in the mitochondria of micu mutants, whereas cytosolic Ca2+ signatures remained unchanged. These findings support the idea that a conserved uniporter system, with composition and regulation distinct from the mammalian machinery, mediates mitochondrial Ca2+ uptake in plants under in vivo conditions. They further suggest that MICU acts as a throttle that controls Ca2+ uptake by moderating influx, thereby shaping Ca2+ signatures in the matrix and preserving mitochondrial homeostasis. Our results open the door to genetic dissection of mitochondrial Ca2+ signaling in plants
