Plant annexins are ubiquitous, soluble proteins capable of Ca²⁺-dependent and Ca²⁺-independent binding to endomembranes and the plasma membrane. Some members of this multigene family are capable of binding to F-actin, hydrolysing ATP and GTP, acting as peroxidases or cation channels. These multifunctional proteins are distributed throughout the plant and throughout the life cycle. Their expression and intracellular localization are under developmental and environmental control. The in vitro properties of annexins and their known, dynamic distribution patterns suggest that they could be central regulators or effectors of plant growth and stress signalling. Potentially, they could operate in signalling pathways involving cytosolic free calcium and reactive oxygen species.

Summary: Hydrogen peroxide is an important regulatory agent in plants. This study demonstrates that exogenous H₂O₂ application to Arabidopsis thaliana root epidermis results in dose‐dependent transient increases in net Ca²⁺ influx. The magnitude and duration of the transients were greater in the elongation zone than in the mature epidermis. In both regions, treatment with the cation channel blocker Gd³⁺ prevented H₂O₂‐induced net Ca²⁺ influx, consistent with application of exogenous H₂O₂ resulting in the activation of plasma membrane Gd³⁺‐sensitive Ca²⁺‐influx pathways. Application of 10 mm H₂O₂ to the external plasma membrane face of elongation zone epidermal protoplasts resulted in the appearance of a hyperpolarization‐activated Ca²⁺‐permeable conductance. This conductance differed from that previously characterized as being responsive to extracellular hydroxyl radicals. In contrast, in mature epidermal protoplasts a plasma membrane hyperpolarization‐activated Ca²⁺‐permeable channel was activated only when H₂O₂ was present at the intracellular membrane face. Channel open probability increased with intracellular [H₂O₂] and at hyperpolarized voltages. Unitary conductance decreased thus: Ba²⁺ > Ca²⁺ (14.5 pS) > Mg²⁺ > Zn²⁺ (20 mm external cation, 1 mm H₂O₂). Lanthanides and Zn²⁺ (but not TEA⁺) suppressed the open probability without affecting current amplitude. The results suggest spatial heterogeneity and differential sensitivity of Ca²⁺ channel activation by reactive oxygen species in the root that could underpin signalling.

Roots are subjected to a range of abiotic stresses as they forage for water and nutrients. Cytosolic free calcium is a common second messenger in the signalling of abiotic stress. In addition, roots take up calcium both as a nutrient and to stimulate exocytosis in growth. For calcium to fulfil its multiple roles must require strict spatio-temporal regulation of its uptake and efflux across the plasma membrane, its buffering in the cytosol and its sequestration or release from internal stores. This prompts the question of how specificity of signaling output can be achieved against the background of calcium’s other uses. Threats to agriculture such as salinity, water availability and hypoxia are signalled through calcium. Nutrient deficiency is also emerging as a stress that is signaled through cytosolic free calcium, with progress in potassium, nitrate and boron deficiency signaling now being made. Heavy metals have the capacity to trigger or modulate root calcium signaling depending on their dose and their capacity to catalyse production of hydroxyl radicals. Mechanical stress and cold stress can both trigger an increase in root cytosolic free calcium, with the possibility of membrane deformation playing a part in initiating the calcium signal. This review addresses progress in identifying the calcium transporting proteins (particularly channels such as annexins and cyclic nucleotide-gated channels) that effect stress-induced calcium increases in roots and explores links to reactive oxygen species, lipid signalling, and the unfolded protein response.

Summary: Extracellular ATP regulates higher plant growth and adaptation. The signalling events may be unique to higher plants, as they lack animal purinoceptor homologues. Although it is known that plant cytosolic free Ca²⁺can be elevated by extracellular ATP, the mechanism is unknown. Here, we have studied roots ofArabidopsis thalianato determine the events that lead to the transcriptional stress response evoked by extracellular ATP. Root cell protoplasts were used to demonstrate that signalling to elevate cytosolic free Ca²⁺is determined by ATP perception at the plasma membrane, and not at the cell wall. Imaging revealed that extracellular ATP causes the production of reactive oxygen species in intact roots, with the plasma membrane NADPH oxidase AtRBOHC being the major contributor. This resulted in the stimulation of plasma membrane Ca²⁺‐permeable channels (determined using patch‐clamp electrophysiology), which contribute to the elevation of cytosolic free Ca²⁺. Disruption of this pathway in theAtrbohCmutant impaired the extracellular ATP‐induced increase in reactive oxygen species (ROS), the activation of Ca²⁺channels, and the transcription of the MAP kinase3 gene that is known to be involved in stress responses. This study shows that higher plants, although bereft of purinoceptor homologues, could have evolved a distinct mechanism to transduce the ATP signal at the plasma membrane.

Plant cell growth and stress signaling require Ca²⁺ influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH^•. In root cells, extracellular OH^• activates a plasma membrane Ca²⁺-permeable conductance that permits Ca²⁺ influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca²⁺-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH^•-activated Ca²⁺- and K⁺-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca²⁺ in response to OH^•. An OH^•-activated Ca²⁺ conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca²⁺-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca²⁺ in plants.

The hydroxyl radical (OH^•) is the most potent yet short-lived of the reactive oxygen species (ROS) radicals. Just as hydrogen peroxide was once considered to be simply a deleterious by-product of oxidative metabolism but is now acknowledged to have signalling roles in plant cells, so evidence is mounting for the hydroxyl radical as being more than merely an agent of destruction. Its oxidative power is harnessed to facilitate germination, growth, stomatal closure, reproduction, the immune response, and adaptation to stress. It features in plant cell death and is a key tool in microbial degradation of plant matter for recycling. Production of the hydroxyl radical in the wall, at the plasma membrane, and intracellularly is facilitated by a range of peroxidases, superoxide dismutases, NADPH oxidases, and transition metal catalysts. The spatio-temporal activity of these must be tightly regulated to target substrates precisely to the site of radical production, both to prevent damage and to accommodate the short half life and diffusive capacity of the hydroxyl radical. Whilst research has focussed mainly on the hydroxyl radical’s mode of action in wall loosening, studies now extend to elucidating which proteins are targets in signalling systems. Despite the difficulties in detecting and manipulating this ROS, there is sufficient evidence now to acknowledge the hydroxyl radical as a potent regulator in plant cell biology.