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 [1–4]. During plant growth and development, eATP is involved in maintaining cell viability, regulating growth rate and direction of some vegetative organs (roots and hypocotyls) [5–9], and regulating reproductive processes [10, 11]. eATP is also involved in regulating stomatal movement and gravitropism [12–15]. 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” [1, 16–22]. As the main eATP hydrolyzing enzyme, extracellular apyrase is involved in terminating signal transduction and maintaining eATP level [23, 24].
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 [21, 25]. 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 [7, 14, 26], NADPH oxidase [12, 14, 27], and ion channels [27–31] 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 [7, 9, 14, 27, 32–34]. After eATP stimulation, altered gene expression and protein synthesis had been observed and proposed to change plant growth & development in response to environmental signals [8, 25, 35].
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 [36–38]. Annexins are involved in seed germination and early seedling growth [39–41], the transition from the vegetative to the reproductive phase [42], pollen germination and tube growth [43], 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 [39, 40, 44–49].
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 [50–53]; 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 [54, 55]. 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 [41, 43, 56, 57]. Annexins have also been observed in the nucleus where they are thought to regulate gene expression [58, 59].
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 [60, 61]. Auxin transporters, especially PIN-FORMED transporters (PINs), play key roles in polar auxin transport [61–63]. 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 [64–68]. Small G protein- or clathrin-mediated vesicle trafficking is involved in PIN trafficking during photo- and gravity-tropic bending growth [69–72]. Most recently, two SNARE proteins were reported to be involved in auxin regulated seedling growth via regulating subcellular trafficking of auxin transporters [68]. 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 [66, 67, 73]. 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 [60–62, 64].
eATP regulates auxin accumulation and 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 [7, 8, 74]. However, the mechanism underlying eATP-regulated PINs abundance and re-location, which in turn alters auxin accumulation and 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 accumulation and distribution in seedlings of annexin-null mutants. Since AtANN3 mutants responded to eATP significantly differently from wildtype, the role of AtANN3 was intensively investigated.