Numerous signal molecules exist in the apoplastic matrix that are responsible for modulating cell metabolism, making the apoplast (including the cell wall and intercellular spaces) an essential modulator of plant cell growth and development. Among these signaling molecules, eATP plays an essential role. Intracellular ATP can leak through plasma membrane (PM) wounds, be secreted in secretory vesicles, or be released through specialized PM transporters (Cao et al. 2014; Roux and Steinebrunner 2007; Tanaka et al. 2010; Pietrowska-Borek et al. 2020). During plant growth and development, eATP is involved in maintaining cell viability, regulating growth rate and direction of some vegetative organs (roots and hypocotyls) (Kim et al. 2006; Yang et al. 2015; Zhu et al. 2017; Zhu et al. 2020; Tonon et al. 2010), and regulating reproductive processes (Wu et al. 2018; Reichler et al. 2009). eATP is also involved in regulating stomatal movement and gravitropism (Chen et al. 2017; Clark et al. 2011; Hao et al. 2012; Tang et al. 2003). In response to biotic or abiotic stresses (e.g. cold stress, salt stress, and pathogen attack), ATP secretion can increase, producing an eATP-stimulated defensive or tolerant responses that act as a “danger signal” (Cao et al. 2014; Chen et al. 2021; Choi et al. 2014b; Deng et al. 2015; Jewell et al. 2019; Kumar et al. 2020; Pham et al. 2020; Tripathi and Tanaka 2018). As the main eATP hydrolyzing enzyme, extracellular apyrase is involved in terminating signal transduction and maintaining eATP level (Clark and Roux 2011; Lim et al. 2014).
To elucidate the mechanisms underlying eATP function, signal transduction of eATP has been extensively investigated over the past two decades. The first step of the eATP signaling pathway is the binding of eATP to its receptors. Two lectin-receptor kinases, P2K1 and P2K2, were identified in Arabidopsis thaliana and shown to be eATP receptors (Choi et al. 2014a; Pham et al. 2020). The two P2K receptors have been shown to participate plant immune responses alone or cooperatively. Several signaling proteins in the PM, including heterotrimeric G proteins (Hao et al. 2012; Zhu et al. 2017; Weerasinghe et al. 2009), NADPH oxidase (Chen et al. 2017; Demidchik et al. 2009; Hao et al. 2012), and ion channels (Demidchik et al. 2009; Shang et al. 2009; Demidchik et al. 2011; Wang et al. 2019; Wang et al. 2018) have been reported to be involved in eATP-stimulated physiological responses. These signal transducers are speculated to be governed directly or indirectly by eATP receptors and involved in eATP-stimulated generation of secondary messengers (e.g. Ca2+, nitric oxide or reactive oxygen species) or intracellular signal transducing cascades (Demidchik et al. 2009; Clark and Roux 2018; Hao et al. 2012; Zhu et al. 2017; Song et al. 2006; Tonon et al. 2010; Wu and Wu 2008). After eATP stimulation, altered gene expression and protein synthesis had been observed and proposed to change plant growth & development in response to environmental signals (Chivasa et al. 2011; Zhu et al. 2020; Choi et al. 2014a).
Annexins are Ca2+- and phospholipid-binding proteins that are located in the PM or inner membrane system, as well as in the cytoplasm of plant cells, that play multiple roles in plant growth, development, and stress responses (Konopka-Postupolska and Clark 2017; Davies 2014; Clark et al. 2012). Annexins are involved in seed germination and early seedling growth (Chu et al. 2012; Cantero et al. 2006; Clark et al. 2005), the transition from the vegetative to the reproductive phase (Lichocka et al. 2018), pollen germination and tube growth (Zhu et al. 2014), etc. Biotic stresses (fungal or viral pathogen attack) and abiotic stresses (cold, heat, salt, and drought) trigger or increase the expression of annexins in various plant species. The expression level of most annexins is positively correlated with plant cells’ tolerance to or defense against stresses (Cantero et al. 2006; Jami et al. 2012; He et al. 2019; Chu et al. 2012; Qiao et al. 2015; Zhang et al. 2011; Yadav et al. 2016; Li et al. 2019).
Annexins are multi-functional proteins that are implicated in Ca2+ signaling, enzymatic metabolic reactions, and vesicle trafficking. Some annexins in maize and Arabidopsis have been shown to build reactive oxygen species-responsive Ca2+ or K+ channels (Laohavisit et al. 2009; Laohavisit et al. 2012; Kodavali et al. 2013; Mu and Zhou 2019); these annexins are thought to be involved in stress-induced Ca2+ signaling. Some annexins showed enzymatic activity, including ATPase, GTPase, and nucleotide phosphodiesterase activity. The peroxidase activity of certain annexins has been shown to be involved in cellular redox reactions: when plants are exposed to stresses, annexins may suppress ROS accumulation, reduce lipid peroxidation, and protect cell activity (Gorecka et al. 2005; Dalal et al. 2014). Annexins are membrane lipid or cytoskeleton binding proteins which localize to the PM and inner membrane where they participate in cytoplasmic vesicle trafficking and cell secretion (Clark et al. 2005; Konopka-Postupolska 2007; Scheuring et al. 2011; Zhu et al. 2014). Annexins have also been observed in the nucleus where they are thought to regulate gene expression (Jami et al. 2008; Huh et al. 2010).
As a multi-functional plant hormone, auxin plays essential roles in regulating growth and development. Plants responding to external stimuli (such as light, gravity, water, etc.) exhibit altered growth rate and orientation. Auxin accumulation and asymmetric distribution are responsible for regulating the elongation rate of plant cells in different parts of plant organs, which results in bending growth of these organs (Zhang and Friml 2020; Han et al. 2021). Auxin transporters, especially PIN-FORMED transporters (PINs), play key roles in polar auxin transport (Han et al. 2021; Lee et al. 2020; Zhou and Luo 2018). Asymmetric distribution of PINs results in unidirectional auxin transport and asymmetric distribution. After stimulation, the subcellular PIN trafficking and PIN phosphorylation alter the localization of PIN proteins, which will alter auxin transport subsequently (Adamowski and Friml 2015; Narasimhan et al. 2021; Barbosa et al. 2018; Tan et al. 2021; Zhang et al. 2021). Small G protein- or clathrin-mediated vesicle trafficking is involved in PIN trafficking during photo- and gravity-tropic bending growth (Ding et al. 2011; Rakusova et al. 2011; Kleine-Vehn et al. 2010; Narasimhan et al. 2020). Most recently, two SNARE proteins were reported to be involved in auxin regulated seedling growth via regulating subcellular trafficking of auxin transporters (Zhang et al. 2021). In response to endogenous or exogenous stimuli, several amino acids in the central long hydrophilic ring of PINs can be phosphorylated, and the phosphorylation is sufficient to modulate the polar distribution, recycling, and ubiquitin-dependent turnover of PIN proteins (Ganguly et al. 2012; Barbosa et al. 2018; Tan et al. 2021). There are 8 members in PIN family in Arabidopsis thaliana, each has distinct spatial-temporal expression and location profiles. In Arabidopsis thaliana seedlings, PINs-mediated auxin re-distribution play essential roles in tropic response of roots, hypocotyls to various stimuli, including gravity, light, salinity and water (Han et al. 2021; Zhang and Friml 2020; Lee et al. 2020; Adamowski and Friml 2015).
eATP regulates auxin asymmetric distribution in the roots of Arabidopsis thaliana, which alters the growth rate and direction of roots. PIN2 and PIN3 have been reported to be involved in eATP-regulated auxin transport (Liu et al. 2012; Zhu et al. 2017; Zhu et al. 2020). However, the mechanism underlying eATP-regulated PINs abundance and re-location, which in turn alters auxin asymmetric distribution, remains unclear. Some annexins have been shown to be necessary components in eATP signaling. Herein, to elucidate the role of annexins in eATP signaling, we investigated the effects of ATP supplementation on growth and auxin distribution in seedlings of annexin-null mutants. Since two AtANN3 null mutant lines responded to eATP significantly differently from wildtype, the role of AtANN3 was intensively investigated.