ER-located Ca ²⁺ ATPase ACA2 regulates Ca ²⁺ cytoplasmic pool linked to root hair growth in Arabidopsis thaliana

Root hairs (RH) are excellent model systems for studying cell size and polarity since they elongate several hundred-fold their original size. Their tip growth is regulated by both intrinsic and environmental signals and is associated with the existence of a highly controlled cytoplasmic tip Ca²⁺ gradient, whose disruption impairs RH development. The molecular mechanisms underlying the Ca ²⁺ homeostasis fine tuning and the Ca ²⁺ organellar contributions to the cytoplasmic pool remain unclear. In the model plant Arabidopsis thaliana , many efflux routes are present, including those that employ Ca ²⁺ -pumps from the Autoinhibited Ca ²⁺ -ATPase (ACA) family. Here, we identified that the ER localized ACA2, and to a lower extent ACA7, are crucial ACAs required to control RH growth. By using genetically encoded Ca ²⁺ biosensors we showed that Ca ²⁺ -dynamics are compromised in the aca2-2 mutant, having lower cytosolic Ca ²⁺ concentration [Ca ²⁺ ] _cyt and growth rate, showing an altered homeostatic calcium setpoint compared to Col-0. Accordingly, the ACA2 mutation changed the dynamics of [Ca ²⁺ ] _cyt oscillations coupled to growth rate, inducing longer periods and more regular oscillations in the dominant high-frequency range (around 22 s), and slower oscillations (around 1 min) in the low-frequency range. Finally, expression of ACA2 with changes in four putative Ca ²⁺ binding residues (ACA2ΔCa ²⁺ ) failed to rescue the RH growth phenotype in the aca2-2 mutant. Collectively, our findings indicate that ER-localized ACA2 and possibly ACA7 are crucial for modulating cytoplasmic Ca ²⁺ signals, possibly composing a critical part of a negative feedback loop, and their absence leads to impairments in RH cell elongation.