Heterogeneous expression of plasma-membrane-localised OsOSCA1.4 complements osmotic sensing based on hyperosmolality and salt stress in Arabidopsis osca1 mutant

Highlights

• OsOSCA1.4 was exclusively localised in the plasma membrane in Arabidopsis mesophyll protoplasts.

• OsOSCA1.4 mediates OICI_cyt and SICI_cyt in HEK293 cells.

• OsOSCA1.4 encodes a calcium-permeable channel.

• Overexpression of OsOSCA1.4 in osca1 complemented osmotic Ca²⁺ signalling in response to hyperosmolality and salt stresses.

Abstract

In plants, both hyperosmolality and salt stress induce cytosolic calcium increases within seconds, referred to as the hyperosmolality-induced [Ca²⁺]_cyt increases, OICI_cyt, and salt stress-induced [Ca²⁺]_cyt increases, SICI_cyt. Previous studies have shown that Arabidopsis reduced hyperosmolality-induced [Ca²⁺]_i increase 1 (OSCA1.1) encodes a hyperosmolality-gated calcium-permeable channel that mediates OICI_cyt in guard cells and root cells. Multiple OSCA members exist in plants; for example, Oryza sativa has 11 OsOSCAs genes, indicating that OSCAs have diverse biological functions. Here, except for OsOSCA4.1, ten full-length OsOSCAs were separately subcloned, in which OsOSCA1.4 was exclusively localised to the plasma membrane and other nine OsOSCAs-eYFP co-localised with an endoplasmic reticulum marker in Arabidopsis mesophyll protoplasts. OsOSCA1.4 was further identified as a calcium-permeable ion channel that activates an inward current after receiving an osmotic signal exerted by hyperosmolality or salt stress, and mediates OICI_cyt and SICI_cyt in human embryonic kidney 293 (HEK293) cells. Moreover, overexpression of OsOSCA1.4 in Arabidopsis osca1 mutant complemented osmotic Ca²⁺ signalling, root growth, and stomatal movement in response to hyperosmolality and salt stress. These results will facilitate further study of OsOSCA-mediated calcium signalling and its distinct roles in rice growth and development.

Introduction

Drought and salt stresses are the two major abiotic constraints on plant growth and development and crop production worldwide [1,2]. After sensing drought and salt stress signals, plants initiate diverse signalling cascades that activate or inhibit the expression of a particular set of genes to modulate metabolism and physiology [3,4]. The first phase common to drought and salt stresses is hyperosmotic stress [2]. In addition, salt stress exerts ionic or ion-toxicity effects on cells. Previous studies have shown that the initial responses of plants to hyperosmolality and salt stress include a rapid increase in cytosolic [Ca²⁺]_cyt (hyperosmolality-induced cytosolic [Ca²⁺] increase, OICI_cyt, and salt stress-induced cytosolic [Ca²⁺] increase, SICI_cyt) [5,6]. Moreover, hyperosmolality and salt stress can induce increases in nucleosolic [Ca²⁺] (OICI_nuc and SICI_nuc) that are independent of OICI_cyt and SICI_cyt in Arabidopsis root cells [6]. Therefore, understanding calcium-involved osmosensing cascades in plants is essential.

Osmotic stress, similar to mechanical stress, affects membrane stretching and membrane–cell wall interactions, which are sensed by mechanosensitive (MS) channels in non-plant systems, including large- and small-conductance MS channels (MscL and MscS, respectively) in bacteria [7]; degenerin/epithelial sodium channels (DEG/ENaC), transient receptor potential (TRP) channels, two-pore domain potassium (K2P) channels, and Piezo channels in mammals [8]; and TRP channels in Drosophila [9,10]. Plants lack TRP and DEG/ENaC genes, but possess a family of mscS-like proteins, comprising two MCA homologues and one Piezo homologue that have been identified as osmosensors [11]. Furthermore, Yuan et al. [12] showed that Arabidopsis reduced hyperosmolality-induced [Ca²⁺]_i increase 1 (OSCA1.1) encodes a hyperosmolality-gated calcium-permeable channel that functions as a osmosensor in plants. In addition, another member of the OSCA family, OSCA1.2/calcium permeable stress-gated cation channel 1 (AtCSC1) could be activated by hyperosmotic shock, via 250 mM NaCl and 500 mM mannitol, in AtCSC1-expressing oocytes [13]. Recently, three groups separately characterised the atomic structures of OSCA1.1, OSCA1.2, and OSCA3.1 using cryo-electron microscopy (cryo-EM), and found that OSCA proteins belong to a new class of MS ion channels with structural similarities to mammalian TMEM16-family proteins [[14], [15], [16]]. In addition, multiple OSCA family members have been found in the genomes of Arabidopsis [12], rice [17], maize [18], and pear [19], indicating that OSCAs play diverse roles in the perception of abiotic stress signals and regulation of stress-responsive gene expression in plants.

In this study, using a heterogeneous expression system, we showed that only OsOSCA1.4 is predominantly localised in the plasma membrane (PM), whereas nine other OsOSCAs are localised mainly in the endoplasmic reticulum (ER) of Arabidopsis mesophyll protoplasts. OsOSCA1.4 encoded a calcium-permeable channel that mediates OICI_cyt and SICI_cyt in HEK293 cells. Furthermore, overexpression o OsOSCA1.4 complemented OICI_cyt and SICI_cyt, root growth, and stomatal movement in response to hyperosmolality and salt stress in Arabidopsis osca1 mutant.