Plant Ca<Superscript>2+</Superscript>-ATPases

Plant calcium pumps, similarly to animal Ca²⁺ pumps, belong to the superfamily of P-type ATPase comprising also the plasma membrane H⁺-ATPase of fungi and plants, Na⁺/K⁺ ATPase of animals and H⁺/K⁺ ATPase of mammalian gastric mucosa. According to their sensitivity to calmodulin the plant Ca²⁺-ATPases have been divided into two subgroups: type IIA (homologues of animal SERCA) and type IIB (homologues of animal PMCA). Regardless of the similarities in a protein sequence, the plant Ca²⁺ pumps differ from those in animals in their cellular localization, structure and sensitivity to inhibitors. Genomic investigations revealed multiplicity of plant Ca²⁺-ATPases; they are present not only in the plasma membranes and ER but also in membranes of most of the cell compartments, such as vacuole, plastids, nucleus or Golgi apparatus. Studies using yeast mutants made possible the functional and biochemical characterization of individual plant Ca²⁺-ATMPases. Plant calcium pumps play an essential role in signal transduction pathways, they are responsible for the regulation of [Ca²⁺] in both cytoplasm and endomembrane compartments. These Ca²⁺-ATPases appear to be involved in plant adaptation to stress conditions, like salinity, chilling or anoxia.     

Simultaneous imaging of ER and cytosolic Ca2+ dynamics reveals long-distance ER Ca2+ waves in plants

Calcium ions (Ca²⁺) play a key role in cell signaling across organisms. In plants, a plethora of environmental and developmental stimuli induce specific Ca²⁺ increases in the cytosol as well as in different cellular compartments including the endoplasmic reticulum (ER). The ER represents an intracellular Ca²⁺ store that actively accumulates Ca²⁺ taken up from the cytosol. By exploiting state-of-the-art genetically encoded Ca²⁺ indicators, specifically the ER-GCaMP6-210 and R-GECO1, we report the generation and characterization of an Arabidopsis (Arabidopsis thaliana) line that allows for simultaneous imaging of Ca²⁺ dynamics in both the ER and cytosol at different spatial scales. By performing analyses in single cells, we precisely quantified (1) the time required by the ER to import Ca²⁺ from the cytosol into the lumen and (2) the time required to observe a cytosolic Ca²⁺ increase upon the pharmacological inhibition of the ER-localized P-Type IIA Ca²⁺-ATPases. Furthermore, live imaging of mature, soil-grown plants revealed the existence of a wounding-induced, long-distance ER Ca²⁺ wave propagating in injured and systemic rosette leaves. This technology enhances high-resolution analyses of intracellular Ca²⁺ dynamics at the cellular level and in adult organisms and paves the way to develop new methodologies aimed at defining the contribution of subcellular compartments in Ca²⁺ homeostasis and signaling.

Dual color imaging allows the simultaneous analysis of calcium dynamics in the endoplasmic reticulum and cytosol from single cells to adult entire plants.     

Molecular aspects of higher plant P-type Ca(2+)-ATPases

Recent genomic data in the model plant Arabidopsis thaliana reveal the existence of at least 11 Ca(2+)-ATPase genes, and an analysis of expressed sequence tags suggests that the number of calcium pumps in this organism might be even higher. A phylogenetic analysis shows that 11 Ca(2+)-ATPases clearly form distinct groups, type IIA (or ECA for ER-type Ca(2+)-ATPase) and type IIB (ACA for autoinhibited Ca(2+)-ATPase). While plant IIB calcium pumps characterized so far are localized to internal membranes, their animal homologues are exclusively found in the plasma membrane. However, Arabidopsis type IIB calcium pump isoforms ACA8, ACA9 and ACA10 form a separate outgroup and, based on the high molecular masses of the encoded proteins, are good candidates for plasma membrane bound Ca(2+)-ATPases. All known plant type IIB calcium ATPases seem to employ an N-terminal calmodulin-binding autoinhibitor. Therefore it appears that the activity of type IIB Ca(2+)-ATPases in plants and animals is controlled by N-terminal and C-terminal autoinhibitory domains, respectively. Possible functions of plant calcium pumps are described and - beside second messenger functions directly linked to calcium homeostasis - new data on a putative involvement in secretory and salt stress functions are discussed.     

Transport,functions, and interaction of calcium and manganese in plant organellar compartments

Calcium (Ca²⁺) and manganese (Mn²⁺) are essential elements for plants and have similar ionic radii and binding coordination. They are assigned specific functions within organelles, but share many transport mechanisms to cross organellar membranes. Despite their points of interaction, those elements are usually investigated and reviewed separately. This review takes them out of this isolation. It highlights our current mechanistic understanding and points to open questions of their functions, their transport, and their interplay in the endoplasmic reticulum (ER), vesicular compartments (Golgi apparatus, trans-Golgi network, pre-vacuolar compartment), vacuoles, chloroplasts, mitochondria, and peroxisomes. Complex processes demanding these cations, such as Mn²⁺-dependent glycosylation or systemic Ca²⁺ signaling, are covered in some detail if they have not been reviewed recently or if recent findings add to current models. The function of Ca²⁺ as signaling agent released from organelles into the cytosol and within the organelles themselves is a recurrent theme of this review, again keeping the interference by Mn²⁺ in mind. The involvement of organellar channels [e.g. glutamate receptor-likes (GLR), cyclic nucleotide-gated channels (CNGC), mitochondrial conductivity units (MCU), and two-pore channel1 (TPC1)], transporters (e.g. natural resistance-associated macrophage proteins (NRAMP), Ca²⁺ exchangers (CAX), metal tolerance proteins (MTP), and bivalent cation transporters (BICAT)], and pumps [autoinhibited Ca²⁺-ATPases (ACA) and ER Ca²⁺-ATPases (ECA)] in the import and export of organellar Ca²⁺ and Mn²⁺ is scrutinized, whereby current controversial issues are pointed out. Mechanisms in animals and yeast are taken into account where they may provide a blueprint for processes in plants, in particular, with respect to tunable molecular mechanisms of Ca²⁺ versus Mn²⁺ selectivity.

Calcium and manganese play essential roles in organellar compartments, yet they share many transport mechanisms, implying synergistic, and antagonistic interactions of both cations.     

A Mathematical Study of the Differential Effects of Two SERCA Isoforms on Ca2+ Oscillations in Pancreatic Islets

Cytosolic Ca²⁺ dynamics are important in the regulation of insulin secretion from the pancreatic β-cells within islets of Langerhans. These dynamics are sculpted by the endoplasmic reticulum (ER), which takes up Ca²⁺ when cytosolic levels are high and releases it when cytosolic levels are low. Calcium uptake into the ER is through sarcoendoplasmic reticulum Ca²⁺-ATPases, or SERCA pumps. Two SERCA isoforms are expressed in the β-cell: the high Ca²⁺ affinity SERCA2b pump and the low affinity SERCA3 pump. Recent experiments with islets from SERCA3 knockout mice have shown that the cytosolic Ca²⁺ oscillations from the knockout islets are characteristically different from those of wild type islets. While the wild type islets often exhibit compound Ca²⁺ oscillations, composed of fast oscillations superimposed on much slower oscillations, the knockout islets rarely exhibit compound oscillations, but produce slow (single component) oscillations instead. Using mathematical modeling, we provide an explanation for this difference. We also investigate the effect that SERCA2b inhibition has on the model β-cell. Unlike SERCA3 inhibition, we demonstrate that SERCA2b inhibition has no long-term effect on cytosolic Ca²⁺ oscillations unless a store-operated current is activated.     

Proton and calcium pumping P-type ATPases and their regulation of plant responses to the environment

Plant plasma membrane H⁺-ATPases and Ca²⁺-ATPases maintain low cytoplasmic concentrations of H⁺ and Ca²⁺, respectively, and are essential for plant growth and development. These low concentrations allow plasma membrane H⁺-ATPases to function as electrogenic voltage stats, and Ca²⁺-ATPases as “off” mechanisms in Ca²⁺-based signal transduction. Although these pumps are autoregulated by cytoplasmic concentrations of H⁺ and Ca²⁺, respectively, they are also subject to exquisite regulation in response to biotic and abiotic events in the environment. A common paradigm for both types of pumps is the presence of terminal regulatory (R) domains that function as autoinhibitors that can be neutralized by multiple means, including phosphorylation. A picture is emerging in which some of the phosphosites in these R domains appear to be highly, nearly constantly phosphorylated, whereas others seem to be subject to dynamic phosphorylation. Thus, some sites might function as major switches, whereas others might simply reduce activity. Here, we provide an overview of the relevant transport systems and discuss recent advances that address their relation to external stimuli and physiological adaptations.

The regulation of plasma membrane H^+-ATPases and autoinhibited Ca2^+-ATPases exhibits a complex and dynamic network of posttranslational regulation. The regulation of plasma membrane H⁺-ATPases and autoinhibited Ca²⁺-ATPases exhibits a complex and dynamic network of posttranslational regulation.

P-type ATPases are found in all domains of life and constitute a large superfamily of membrane-bound pumps that share a common machinery, including a reaction cycle that involves catalytic phosphorylation of an Asp, resulting in a phosphorylated intermediate (reviewed in Palmgren and Nissen, 2011; (hence the name P-type; Box 1). The catalytic phosphoryl-aspartate intermediate is not to be confused with regulatory phosphorylation, which occurs on Ser, Thr, and Tyr residues. Five major families of P-type ATPases have been characterized (P1–5), each of which is divided into a number of subfamilies (named with letters). Plasma membrane H⁺-ATPases are classified as P3A ATPases, whereas Ca²⁺ pumps constitute P2A and P2B ATPases. In plants, these pumps are best characterized in the model plant Arabidopsis thaliana (Arabidopsis).Box 1Enzymology of P-type ATPases.P-type ATPases (reviewed in Palmgren and Nissen, 2011) alternate between two extreme conformations during their catalytic cycle: a high-affinity (with respect to ATP and the ion to be exported) Enzyme1 (E1) state, and a low-affinity Enzyme2 (E2) state. Many P-type ATPases are autoinhibited by built-in molecular constraints, namely their C- and N-terminal (for plasma membrane H⁺-ATPases; Palmgren et al., 1999) or N-terminal (for P2B Ca²⁺-ATPases; Malmström et al., 1997) regulatory (R) domains of approximately 100 amino acid residues, which act as brakes by stabilizing the pumps in a low-affinity conformation (Palmgren and Nissen, 2011), most likely E2. Neutralizing the R domain results in a shift in conformational equilibrium towards a high-affinity state, likely E1. In this way, the R domains of plasma membrane H⁺-ATPases and Ca²⁺-ATPases allow posttranslational modification events to control the turnover numbers of these pumps. A structure of a plasma membrane H⁺-ATPase (from the distantly related yeast S. cerevisiae) in its autoinhibited state has been solved (Heit et al., 2021). Its R domain is situated adjacent to the P domain, which would suggest that the R domain functions to restrict the conformational flexibility of the pump. Normally, the hydrolysis of ATP and transport are tightly coupled in P-type ATPases. Therefore, P-type ATPases hydrolyze bound ATP as soon as their ligand-binding site(s) in the membrane region are occupied, but not before. Thus, increasing the ligand affinity of an ATPase simultaneously increases its turnover number, provided that the concentration of ATP is not limiting, which is rarely the case in cells. A specific feature of plasma membrane H⁺-ATPases is that in the autoinhibited state, ATP hydrolysis is only loosely coupled to H⁺ pumping, whereas pump activation results in tight coupling, with one H⁺ pumped per ATP split (Pedersen et al., 2018).In response to internal and/or external cues, plasma membrane H⁺-ATPase and Ca²⁺-ATPase activities are controlled by intracellular concentrations of H⁺ and Ca²⁺, respectively, via interacting proteins, through posttranslational modification by phosphorylation, and by regulated trafficking of the pump to and from the plasma membrane. Their regulation sometimes involves changes in gene expression and turnover, although this is rare, perhaps because both processes are time- and energy-consuming (Haruta et al., 2018).     

Regulation of PKC Mediated Signaling by Calcium during Visceral Leishmaniasis

Calcium is an ubiquitous cellular signaling molecule that controls a variety of cellular processes and is strictly maintained in the cellular compartments by the coordination of various Ca²⁺ pumps and channels. Two such fundamental calcium pumps are plasma membrane calcium ATPase (PMCA) and Sarco/endoplasmic reticulum calcium ATPase (SERCA) which play a pivotal role in maintaining intracellular calcium homeostasis. This intracellular Ca²⁺ homeostasis is often disturbed by the protozoan parasite Leishmania donovani, the causative organism of visceral leishmaniasis. In the present study we have dileneated the involvement of PMCA4 and SERCA3 during leishmaniasis. We have observed that during leishmaniasis, intracellular Ca²⁺ concentration was up-regulated and was further controlled by both PMCA4 and SERCA3. Inhibition of these two Ca²⁺-ATPases resulted in decreased parasite burden within the host macrophages due to enhanced intracellular Ca²⁺. Contrastingly, on the other hand, activation of PMCA4 was found to enhance the parasite burden. Our findings also highlighted the importance of Ca²⁺ in the modulation of cytokine balance during leishmaniasis. These results thus cumulatively suggests that these two Ca²⁺-ATPases play prominent roles during visceral leishmaniasis.     

A comparison of sarcoplasmic reticulum function in fast and slow skeletal muscle using crude homogenate and isolated vesicles

Sarcoplasmic reticulum vesicles (FSR) were isolated from relatively homogeneous muscle samples representative of type I, IIA, and IIB fibers and Ca²⁺ uptake (V_max and total capacity) determined. Crude homogenates of these same fiber populations were also assayed for Ca²⁺ uptake and the results compared to the FSR values. Both techniques produced qualitatively similar results and demonstrated distinct fiber type differences in both the rate and extent of Ca²⁺ uptake. The results obtained support the contention that the crude homogenate technique accurately reflects the activity of the sarcoplasmic reticulum.     

The effect of exercise-training on sarcoplasmic reticulum function in fast and slow skeletal muscle

The effect of endurance exercise on the capacity of crube homogenates (CH) to sequester Ca²⁺ was determined in the slow type I soleus, the fast type IIA deep region of the vastus lateralis (DVL), and the fast type IIB superficial region of the vastus lateralis (SVL). The Ca²⁺ uptake capacity was affected by exercise in a fiber type specific manner. The fast-twitch SVL showed a 35% decrease in the maximal rate of Ca²⁺ uptake (V_max) and a significantly lower K_m while the slow soleus and fast DVL were unaltered. The time course of Ca²⁺ uptake, and the peak amount of Ca²⁺ sequestered was not altered by exercise in any of the muscles studied. The homogenates from the exercise-trained soleus muscles exhibited an increased ability to retain Ca²⁺ and in this capacity became more like fast muscle.     

Vacuolar Ca(2+) uptake

Calcium transporters that mediate the removal of Ca²⁺ from the cytosol and into internal stores provide a critical role in regulating Ca²⁺ signals following stimulus induction and in preventing calcium toxicity. The vacuole is a major calcium store in many organisms, particularly plants and fungi. Two main pathways facilitate the accumulation of Ca²⁺ into vacuoles, Ca²⁺-ATPases and Ca²⁺/H⁺ exchangers. Here I review the biochemical and regulatory features of these transporters that have been characterised in yeast and plants. These Ca²⁺ transport mechanisms are compared with those being identified from other vacuolated organisms including algae and protozoa. Studies suggest that Ca²⁺ uptake into vacuoles and other related acidic Ca²⁺ stores occurs by conserved mechanisms which developed early in evolution.     

The origin and function of calmodulin regulated Ca2+ pumps in plants

While Ca²⁺ signaling plays an important role in both plants and animals, the machinery that codes and decodes these signals have evolved to show interesting differences and similarities. For example, typical plant and animal cells both utilize calmodulin (CaM)-regulated Ca²⁺ pumps at the plasma membrane to help control cytoplasmic Ca²⁺ levels. However, in flowering plants this family of pumps has evolved with a unique structural arrangement in which the regulatory domain is located at the N-terminal instead of C-terminal end. In addition, some of the plant isoforms have evolved to function at endomembrane locations. For the 14 Ca²⁺ pumps present in the model plant Arabidopsis, molecular genetic analyses are providing exciting insights into their function in diverse aspects of plant growth and development.     

Three-dimensional spatio-temporal modelling of store operated Ca2+ entry: Insights into ER refilling and the spatial signature of Ca2+ signals

The spatial organisation of Orai channels and SERCA pumps within ER-PM junctions is important for enhancing the versatility and specificity of sub-cellular Ca²⁺ signals generated during store operated Ca²⁺ entry (SOCE). In this paper, we present a novel three dimensional spatio-temporal model describing Ca²⁺ dynamics in the ER-PM junction and sub-PM ER during SOCE. We investigate the role of Orai channel and SERCA pump location to provide insights into how these components shape the Ca²⁺ signals generated and affect ER refilling. We find that the organisation of Orai channels within the ER-PM junction controls the amplitude and shape of the Ca²⁺ profile but does not enhance ER refilling. The model shows that ER refilling is only weakly affected by the location of SERCA2b pumps within the ER-PM junction and that the placement of SERCA2a pumps within the ER-PM junction has much greater control over ER refilling.     

A tomato ER-type Ca-ATPase, LCA1, has a low thapsigargin-sensitivity and can transport manganese

Recombinant Ca²⁺-ATPase from tomato (i.e. LCA1 for Lycopersicon esculentum [Since the identification and naming of LCA1, the scientific name for the tomato has been changed to Solanum lycopersicum.] Ca-ATPase) was heterologously expressed in yeast for structure-function characterization. We investigate the differences between plant and animal Ca pumps utilizing comparisons between chicken and rabbit SERCA-type pumps with Arabidopsis (ECA1) and tomato plant (LCA1) Ca²⁺-ATPases. Enzyme function was confirmed by the ability of each Ca²⁺-ATPase to rescue K616 growth on EGTA-containing agar and directly via in vitro ATP hydrolysis. We found LCA1 to be ∼300-fold less sensitive to thapsigargin than animal SERCAs, whereas ECA1 was thapsigargin-resistant. LCA1 showed typical pharmacological sensitivities to cyclopiazonic acid, vanadate, and eosin, consistent with it being a P_IIA-type Ca²⁺-ATPase. Possible amino acid changes responsible for the reduced plant thapsigargin-sensitivity are discussed. We found that LCA1 also complemented K616 yeast growth in the presence of Mn²⁺, consistent with moving Mn²⁺ into the secretory pathway and functionally compensating for the lack of secretory pathway Ca-ATPases (SPCAs) in plants.     

Regulation of Plant Plasma Membrane H<Superscript>+</Superscript>- and Ca<Superscript>2+</Superscript>-ATPases by Terminal Domains

In the last few years, major progress has been made to elucidate the structure, function, and regulation of P-type plasma membrane H⁺-and Ca²⁺-ATPases. Even though a number of regulatory proteins have been identified, many pieces are still lacking in order to understand the complete regulatory mechanisms of these pumps. In plant plasma membrane H⁺- and Ca²⁺-ATPases, autoinhibitory domains are situated in the C- and N-terminal domains, respectively. A model for a common mechanism of autoinhibition is discussed.     

Effects of mechanical and chemical disruption on the ATP-phosphohydrolase activity and ultrastructure of sperm flagella.

The Ca²⁺- and Mg²⁺-ATP-phosphohydrolase (ATPase) activities measurable in suspensions of and extracts from bull epididymal spermatozoa flagella, sonicated for 2 min at 8 W, were compared with those of control flagella after 0, 3, and 18 h dialysis against an alkaline 0.5 M KCl extracting solution. Activity was measured in the absence of and in the presence of oligomycin. The effects of sonication and extraction on structural components within the flagella were visualized electron microscopically. Sonication caused fragmentation of flagella, extensive disruption of mitochondria and an immediate (0 h) increase in both Ca²⁺- and Mg²⁺-ATP-ase activity. Prolonged dialysis resulted in solubilization of specific flagellar structures, partial disruption of mitochondrial integrity, and increases in ATPase activity. Mg²⁺-ATPases of flagellar suspensions and extracts were greater than Ca²⁺-ATPases, and a part of this Mg²⁺-ATPase activity was inhibited by oligomycin. Therefore, Mg²⁺-ATPases from disrupted mitochondria contribute to the Mg²⁺-ATPase activities measurable in suspensions of and extracts from bull sperm flagella. This study emphasizes the necessity of evaluating the effects of the mechanical and chemical treatments used in fractionating cells before interpreting the biochemical information derived from their isolated components.     

Calcium Signaling Network in Plants: An Overview

Calcium ion (Ca²⁺) is one of the very important ubiquitous intracellular second messenger molecules involved in many signal transduction pathways in plants. The cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) have been found to increased in response to many physiological stimuli such as light, touch, pathogenic elicitor, plant hormones and abiotic stresses including high salinity, cold and drought. This Ca²⁺ spikes normally result from two opposing reactions, Ca²⁺ influx through channels or Ca²⁺ efflux through pumps. The removal of Ca²⁺ from the cytosol against its electrochemical gradient to either the apoplast or to intracellular organelles requires energized ‘active’ transport. Ca²⁺-ATPases and H⁺/Ca²⁺ antiporters are the key proteins catalyzing this movement. The increased level of Ca²⁺ is recognised by some Ca²⁺-sensors or calcium-binding proteins, which can activate many calcium dependent protein kinases. These kinases regulate the function of many genes including stress responsive genes, resulted in the phenotypic response of stress tolerance. Calcium signaling is also involved in the regulation of cell cycle progression in response to abiotic stress. The regulation of gene expression by cellular calcium is also crucial for plant defense against various stresses. However, the number of genes known to respond to specific transient calcium signals is limited. This review article describes several aspects of calcium signaling such as Ca²⁺ requiremant and its role in plants, Ca²⁺ transporters, Ca²⁺-ATPases, H⁺/ Ca²⁺-antiporter, Ca²⁺-signature, Ca²⁺-memory and various Ca²⁺-binding proteins (with and without EF hand).Key Words: Calcium binding proteins, Ca²⁺ channel, Ca²⁺-dependent protein kinases, Ca²⁺/H⁺ antiport, calcium memory, calcium sensors, calcium signatures, Ca²⁺-transporters, EF hand motifs, plant signal transduction     

Orai3 channel is the 2-APB-induced endoplasmic reticulum calcium leak

We have studied in HeLa cells the molecular nature of the 2-APB induced ER Ca²⁺ leak using synthetic Ca²⁺ indicators that report changes in both the cytoplasmic ([Ca²⁺]_i) and the luminal ER ([Ca²⁺]_ER) Ca²⁺ concentrations. We have tested the hypothesis that Orai channels participate in the 2-APB-induced ER Ca²⁺ leak that was characterized in the companion paper. The expression of the dominant negative Orai1 E106A mutant, which has been reported to block the activity of all three types of Orai channels, inhibited the effect of 2-APB on the [Ca²⁺]_ER but did not decrease the ER Ca²⁺ leak after thapsigargin (TG). Orai3 channel, but neither Orai1 nor Orai2, colocalizes with expressed IP₃R and only Orai3 channel supported the 2-APB-induced ER Ca²⁺ leak, while Orai1 and Orai2 inhibited this type of ER Ca²⁺ leak. Decreasing the expression of Orai3 inhibited the 2-APB-induced ER Ca²⁺ leak but did not modify the ER Ca²⁺ leak revealed by inhibition of SERCA pumps with TG. However, reducing the expression of Orai3 channel resulted in larger [Ca²⁺]_i response after TG but only when the ER store had been overloaded with Ca²⁺ by eliminating the acidic internal Ca²⁺ store with bafilomycin. These data suggest that Orai3 channel does not participate in the TG-revealed ER Ca²⁺ leak but forms an ER Ca²⁺ leak channel that is limiting the overloading with Ca²⁺ of the ER store.     

Localization of endogenous ATPases at the nerve terminal

We have investigated the localization of a set of intrinsic ATPase activities associated with purified synaptic plasma membranes and consisting of (a) a Mg²⁺-ATPase; (b) an ATPase active at high concentrations of Ca²⁺ in the absence of Mg²⁺ (Ca_H-ATPase); (c) a Ca²⁺ requiring Mg²⁺-dependent ATPase (Ca + Mg)-ATPase, stimulated by calmodulin (Ca-CaM-ATPase); (d) a Ca²⁺-dependent ATPase stimulated by dopamine (DA-ATPase); and (e) the ouabain-sensitive (Na + K)-ATPase. The following results were obtained: (1) All ATPases are largely confined to the presynaptic membrane; (2) the DA-, (Ca + Mg)-, (Ca-CaM)-, and (Na + K)-ATPases are oriented with their ATP hydrolysis sites facing the synaptoplasm; (3) the Mg- and Ca_H-ATPases are oriented with their ATP hydrolysis sites on the junctional side of the presynaptic membrane and are therefore classified as ecto-ATPases of as yet unknown function.