The structural bases for agonist diversity in an Arabidopsis thaliana glutamate receptor-like channel.

Arabidopsis 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.

Phosphate Starvation Alters Abiotic-Stress-Induced Cytosolic Free Calcium Increases in Roots.

Phosphate (Pi) deficiency strongly limits plant growth, and plant roots foraging the soil for nutrients need to adapt to optimize Pi uptake. Ca2+ is known to signal in root development and adaptation but has to be tightly controlled, as it is highly toxic to Pi metabolism. Under Pi starvation and the resulting decreased cellular Pi pool, the use of cytosolic free Ca2+ ([Ca2+]cyt) as a signal transducer may therefore have to be altered. Employing aequorin-expressing Arabidopsis (Arabidopsis thaliana), we show that Pi starvation, but not nitrogen starvation, strongly dampens the [Ca2+]cyt increases evoked by mechanical, salt, osmotic, and oxidative stress as well as by extracellular nucleotides. The altered root [Ca2+]cyt response to extracellular ATP manifests during seedling development under chronic Pi deprivation but can be reversed by Pi resupply. Employing ratiometric imaging, we delineate that Pi-starved roots have a normal response to extracellular ATP at the apex but show a strongly dampened [Ca2+]cyt response in distal parts of the root tip, correlating with high reactive oxygen species levels induced by Pi starvation. Excluding iron, as well as Pi, rescues this altered [Ca2+]cyt response and restores reactive oxygen species levels to those seen under nutrient-replete conditions. These results indicate that, while Pi availability does not seem to be signaled through [Ca2+]cyt, Pi starvation strongly affects stress-induced [Ca2+]cyt signatures. These data reveal how plants can integrate nutritional and environmental cues, adding another layer of complexity to the use of Ca2+ as a signal transducer.

Endoplasmic reticulum-localized CCX2 is required for osmotolerance by regulating ER and cytosolic Ca2+ dynamics in Arabidopsis.

Ca2+ signals in plant cells are important for adaptive responses to environmental stresses. Here, we report that the Arabidopsis CATION/Ca2+ EXCHANGER2 (CCX2), encoding a putative cation/Ca2+ exchanger that localizes to the endoplasmic reticulum (ER), is strongly induced by salt and osmotic stresses. Compared with the WT, AtCCX2 loss-of-function mutant was less tolerant to osmotic stress and displayed the most noteworthy phenotypes (less root/shoot growth) during salt stress. Conversely, AtCCX2 gain-of-function mutants were more tolerant to osmotic stress. In addition, AtCCX2 partially suppresses the Ca2+ sensitivity of K667 yeast triple mutant, characterized by Ca2+ uptake deficiency. Remarkably, Cameleon Ca2+ sensors revealed that the absence of AtCCX2 activity results in decreased cytosolic and increased ER Ca2+ concentrations in comparison with both WT and the gain-of-function mutants. This was observed in both salt and nonsalt osmotic stress conditions. It appears that AtCCX2 is directly involved in the control of Ca2+ fluxes between the ER and the cytosol, which plays a key role in the ability of plants to cope with osmotic stresses. To our knowledge, Atccx2 is unique as a plant mutant to show a measured alteration in ER Ca2+ concentrations. In this study, we identified the ER-localized AtCCX2 as a pivotal player in the regulation of ER Ca2+ dynamics that heavily influence plant growth upon salt and osmotic stress.

Light Sheet Fluorescence Microscopy Quantifies Calcium Oscillations in Root Hairs of Arabidopsis thaliana.

Calcium oscillations play a role in the regulation of the development of tip-growing plant cells. Using optical microscopy, calcium oscillations have been observed in a few systems (e.g. pollen tubes, fungal hyphae and algal rhizoids). High-resolution, non-phototoxic and rapid imaging methods are required to study the calcium oscillation in root hairs. We show that light sheet fluorescence microscopy is optimal to image growing root hairs of Arabidopsis thaliana and to follow their oscillatory tip-focused calcium gradient. We describe a protocol for performing live imaging of root hairs in seedlings expressing the cytosol-localized ratiometric calcium indicator Yellow Cameleon 3.6. Using this protocol, we measured the calcium gradient in a large number of root hairs. We characterized their calcium oscillations and correlated them with the rate of hair growth. The method was then used to screen the effect of auxin on the properties of the growing root hairs.

The EF-Hand Ca2+ Binding Protein MICU Choreographs Mitochondrial Ca2+ Dynamics in Arabidopsis.

Plant organelle function must constantly adjust to environmental conditions, which requires dynamic coordination. Ca(2+) signaling may play a central role in this process. Free Ca(2+) dynamics are tightly regulated and differ markedly between the cytosol, plastid stroma, and mitochondrial matrix. The mechanistic basis of compartment-specific Ca(2+) 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 Ca(2+) uniporter machinery in mammals. MICU binds Ca(2+) and localizes to the mitochondria in Arabidopsis. In vivo imaging of roots expressing a genetically encoded Ca(2+) sensor in the mitochondrial matrix revealed that lack of MICU increased resting concentrations of free Ca(2+) in the matrix. Furthermore, Ca(2+) elevations triggered by auxin and extracellular ATP occurred more rapidly and reached higher maximal concentrations in the mitochondria of micu mutants, whereas cytosolic Ca(2+) signatures remained unchanged. These findings support the idea that a conserved uniporter system, with composition and regulation distinct from the mammalian machinery, mediates mitochondrial Ca(2+) uptake in plants under in vivo conditions. They further suggest that MICU acts as a throttle that controls Ca(2+) uptake by moderating influx, thereby shaping Ca(2+) signatures in the matrix and preserving mitochondrial homeostasis. Our results open the door to genetic dissection of mitochondrial Ca(2+) signaling in plants.