This study integrates all available information from literature to define ABA signalling pathway and presents it in a systems view for clearer understanding of the role of Ca2+ in stomatal closure. Importantly, it incorporates into an Asynchronous Boolean model time delays sourced from an extensive literature search. The model predicted the timing of ABA events and mutant behaviour close to biology. It revealed biologically reported timing for Ca2+ activation and Ca2+ dynamics consistent with biology. It also predicts that Ca2+ elevation is not essential in stomatal closure but it can accelerate closure, consistent with previous findings, but our model further explains that acting as a mediator, Ca2+ accelerates stomatal closure by enhancing plasma membrane slowly activating anion channel SLAC1 and actin rearrangement. It shows statistical significance of Ca2+ induced acceleration of closure and that of Ca2+ induced acceleration of SLAC1 activation. Further, the model demonstrates that Ca2+ enhances resilience of closure to perturbation of important elements; especially, ROS pathway, as did previous ABA model, and even to the ABA signal disruption. It goes further to elucidate the mechanisms by which Ca2+ engenders stomatal closure in these perturbations.Inconsistent hypotheses have proposed Ca Calcium (Ca2+), the most ubiquitous second messenger in plant cells, is tightly connected with all the important functions of ABA signalling  and lipid)  in response to drought invokes a series of cell signalling pathways causing rapid stomatal closure in plants to prevent water loss and cell dehydration2+ in ABA signalling is not yet fully established. Some studies have shown that Ca2+ is fundamental to stomatal closure4. Other studies have shown slower stomatal closure in response to conditions that prevented Ca2+ elevation in guard cell cytoplasm5. Some other studies, however, have reported that Ca2+ elevation is not needed for stomatal closure6 by revealing uninterrupted downstream events, such as ion channel regulation8 (which can be regulated by Ca2+ as well), even after inhibition of Ca2+ signalling in guard cells. Further, some studies have suggested that there may be two transduction routes in ABA induced stomatal closure (Ca2+ dependent and independent) where activation of either pathway is sufficient to induce stomatal closure9. More recently another group of scientists hypothesized that certain threshold level of Ca2+ elevation regulates stomatal closure and a defined pattern of Ca2+ oscillation is needed to inhibit stomatal reopening, or maintain closure3. Supporting this latter hypothesis on closure maintenance, some research has reported that ABA-induced steady state stomatal closure is characterised by a series of Ca2+ oscillations with defined frequency and amplitude3. Together, these hypotheses raise a fundamental question about the functional significance of Ca2+ within the complex ABA signalling in plant guard cells.According to experimental literature, the role of Ca2+ signals within the ABA signalling system in the past years, the regulatory principles that convert cellular Ca2+ signals into guard cell responses remain unexplored10. Engineering the guard cell signal transduction network could therefore make a major contribution to solving the mystery of Ca2+ signalling in guard cells. There have been few modelling attempts to solve this mystery due to the lack of knowledge of biological connectivity between the players of the large ABA network and extreme lack of data. The last modelling attempt of ABA signalling prior to our study, a random order asynchronous Boolean model with 43 proteins and 76 interactions, was reported in 200611. Their model identified the important elements of the ABA network, but the reproducibility of realistic temporal events was limited due to the assumed randomness in network updating order as the model did not incorporate biological time delays associated with the reactions. However, authors concluded that stomatal closure is accelerated when the Ca2+ level is elevated but ABA signalling can achieve the end target even without Ca2+ in the system. In supporting their conclusions, they make the prediction that their model predictions will stay the same as the co-topological properties of the network stay the same even with future network modifications. Nevertheless, they reported on the essentiality of Ca2+ to the system under some important nodal disruptions11. However, later in 2010, the same authors12 acknowledged the significant dependence of the system behaviour on the manner of update indicating that incorporation of a biologically valid updating scheme (timing) may produce biologically realistic behaviour. Therefore, the following major additions different from the above mentioned previous ABA signalling network model11 were introduced to the existing ABA signalling network to better study the system behaviour. First, we incorporated a biologically valid updating scheme (timing) into the model. Second, the network obtained is significantly more comprehensive with the incorporation of a number of important novel experimental findings.Despite the progress in our understanding of the transmission of Ca2+ in stomatal closure, we first created the most complete ABA signalling network at the time of this study (56 nodes and 127 interactions) based on new information discovered since 2006  - elucidating the proper role of the latter is the aim of this study. Two other signalling subsystems, coordinated by the ABA signal, facilitate the above two main functions and these are ROS signalling (pink box) and Lipid Signalling (grey box).We carefully studied all the elements and interactions as well as the logic of operation of the extended ABA signalling pathway shown in Fig. 2+ signalling regulates the level of Ca2+ and connects Ca2+ with osmoregulation, actin rearrangement, lipid and ROS pathways  - have substantially differentiated this extended network topology from the previous Li et al.11 network. The upstream components of the Ca2+ regulatory pathway are nearly similar in both models but the definition of the Ca2+ dependent downstream regulations is considerably different in the two models. For example, Ca2+ release by the internal organelles is regulated by four independent regulators (Inositol trisphosphate (InSP3), Inositol hexaphosphate (InSP6), Cyclic guanosine monophosphate (cGMP) and Cyclic adenosine diphosphoribose (cADPR)) in the Li et al.11 model but we assume that these four regulators are not independent (InSP6 is the phosphorylated product of InSP315 and cGMP enhances the production of cADPR16); therefore, our model considers them as two regulatory paths. Further, our model considers the contribution of structural rearrangements to cytosolic Ca2+ increase through stretch activated Ca2+ channels in the plasma membrane, which was not defined in the previous model. Moreover, the previous model considered the Ca2+ efflux system as a single node regulated by Ca2+. We are certain that the Ca2+ efflux system consists of two effluxes (Ca2+-ATPase17 and H+/Ca2+ exchangers (CAX)18) that are differently regulated by their respective regulators. Therefore, our model considers individual regulations of the two Ca2+ effluxes assuming that the addition of all known regulators will produce biologically evidenced Ca2+ signature with our asynchronous Boolean model incorporating real timing. Further, several novel additions, such as Ca2+ involvement in slowly activating anion channel (SLAC1) regulation and ROS production via Ca2+ dependent protein kinases (CDPK), and Ca2+ regulation of Ca2+-dependent vacuolar malate channel, ATALMT6, as well as actin binding proteins, to our model make it considerably different from Li et al.11 model.Ca2+. Overall, stomatal closure is accompanied by several hierarchical functional events as indicated in the figure: Signal Perception, Osmotic Regulation, Structural Rearrangements, Ca2+ Signalling and Maintenance of Closure. In brief, ABA recognition by its receptor complex activates the SNF-related serine/threonine protein kinase (SnRK2)  in Fig. 2+  . A vari 2+ Fig. . The ABA(D) Fig. ) in para(D) Fig.  until th2+ that constitutes the most connected functional set in ABA signalling  is an evolutionary constraint in all cell types. This is because Ca2+ can be toxic at higher concentrations as higher levels of free cytosolic Ca2+ bind with orthophosphates present in ATP causing precipitation of low soluble calcium salts in the cytoplasm20; this alters the biological reactions demanding free energy transduction. To maintain Ca2+ at a level safe to cellular functions, guard cells employ a well-developed signalling network   ↔ Ca2+ . We collected fragmentary timing data for each interaction from an extensive search through various sources and fitted them into the ABA time scale while minimizing discrepancies. This was considered the best and most realistic option for incorporating timing into the model due to the lack of availability of a complete set of proteomics time series data to order the correct sequence of update in the ABA system. Under this condition, we constructed different but biologically realistic time delays for the 127 interactions in the ABA network.2+ signalling as our aim is to question the existing hypotheses on Ca2+ signals in ABA signalling.We assumed that physical bindings, posttranslational modifications of proteins, ion channel activities, membrane charge alterations and other interactions determine the dynamics of the system. The key role here is played by the associated time delays as they correspond to the actual reaction speeds; for example, activation of a specific target at lower expression level is represented by lower time delay and vice versa. This approach incorporating realistic time delays is the first exploratory step to understand problems which are too complex to model using partial differential equations or other continuous approaches. In this model, the output of a Boolean function representing a reaction is the combined effect of the inputs and their associated time delays, bringing it one step closer to continuous dynamics. The value of this approach is further boosted by the current perception that the actual timing could be more important than the exact levels of proteins.  resulting in different steady state behaviour in the ABA signalling system. Some of the observed patterns are spurious as a result of assumed initial conditions for some nodes due to the lack of knowledge of biologically possible initial protein activity levels as well as currently unknown regulatory mechanisms for some nodes  in the system reach a fixed state with the exception of few nodes (14) that include Ca2+ and Ca2+ governed molecules that form a limit cycle attractor. Stationary nodes which were shared by both models  showed similar steady state levels but the dynamic nodes showed non-identical oscillatory patterns. .Our model further showed that different initial conditions of the system cause behavioural differences in rhythmic Care3 Figs . The pre2+throughout the simulation, we conclude that the system converges into these rhythmic oscillations after the onset of closure. This raises the question as to why the rhythmic oscillations are needed after the onset of closure. Does the system require Ca2+ oscillations to maintain the steady state of stomatal closure? Biologically, if the ABA system does not need any signalling communication after the onset of closure, the steady state dynamics of the system should be a fixed-point attractor. If the ABA system can close stomata with no Ca2+ oscillations and if it can maintain closure with the same steady level activities of the other network elements, then it does not need Ca2+ oscillations to appear in the second phase. If any signalling communication occurs after the onset of closure, i.e. oscillatory behaviour, it should be assigned some important role during the steady state.Observing temporal dynamics of Ca2+ oscillations appear after stomatal closure supports one of the existing hypotheses that there are two phases in ABA induced stomatal closure. The first phase induces closure (rapid phase) and the second phase maintains closure by inhibiting the reopening of the stomata. This hypothesis was supported by experimental literature which alludes to: “…short term ‘calcium-reactive’ closure that occurred rapidly when cytosolic Ca2+ was elevated, and ‘calcium programmed’ long-term steady-state closure that occurred with Ca2+ oscillations with a defined range of frequency, duration and amplitude”3. Accordingly, the role of cytosolic Ca2+ oscillations may be for the second phase (maintenance of closure) to keep the stomata closed without reopening because stomata are liable to open during daytime in response to favourable abiotic signals, such as blue light. However, our current model has not specifically incorporated the relevant pathways that control the inhibition of stomatal reopening to evaluate it further. This is because cell signalling mechanisms for stomatal reopening is another complex signalling system, which comprises nearly 70 molecules connected by 150 interactions governed by four signals . This was recently modelled using a multi-level dynamic model25 showing that there is Ca2+ regulated robust cross-talk between blue light and ABA. Moreover, a recent study done by Minguet-Parramona et al.26 (a model on guard cell ion transport) reported that rhythmic oscillations of Ca2+ are a by-product of ion channel activities in guard cells, specifically AHA1, an active H+ transporter on the plasma membrane that attempts to open stomata27. Further, experimental evidence also suggests that regular Ca2+ oscillations may be more important for the maintenance of closure than for the induction of closure23. Therefore, in this paper we emphasize the potential roles of Ca2+ elevations in short term closure (Ca2+-reactive).The observation that the Ca2+ appears as a hub element in the system, communicating with almost all the other functional sets. Hub components in a scale-free network  are extremely important and therefore have the potential to disturb the functional stability of the network. Importance of Ca2+ elevation prior to the system reaching steady state dynamics was, therefore, evaluated by the probability of stomatal closure . The initial conditions used in the simulation were identical in both systems. The model results revealed that the presence of Ca2+ has no effect on the probability of closure, where the probability (~80%) remains the same in both Ca2+ dependent and independent systems  activates SLAC1 independently of Ca2+. To date, no evidence confirms that activation of SLAC1 in planta is solely through phosphorylation by OST1 as in oocytes. The second hypothesis claims that there is no Ca2+ independent SLAC1 regulation pathway in guard cells. Different research groups supporting the second hypothesis have shown that background level Ca2+ was required for ABA induced SLAC1 anion channel function using planta data34. One study, which claimed the second hypothesis showed that anion currents were observed even after blocking the Ca2+ entry into the cytoplasm but buffering Ca2+ below physiological level abolished ABA activation of anion channels8. In this study, we assumed that guard cell SLAC1 channel can be regulated independently by either OST1 (without elevating Ca2+) or Ca2+ dependent pathways via CPKs. Ca2+ elevation independent OST1 activation of SLAC1 was evidenced in oocyte expression system with plant SLAC1 and OST1 proteins31, hence, it may exist in plant guard cells as well. This and many other observations point to the possibility of Ca2+ independent SLAC1 activation by OST1 but some inconsistent counter evidence exists as explained below.At present, there are two opposing arguments about the essentiality of Ca33 supported Ca2+ dependence of the system by showing altered stomatal sensitivity of quadruple mutants (cpk5/6/11/23) of Ca2+ dependent protein kinases (CPKs) to ABA. Two more studies from the same group35 reported that CPK6 and CPK23 display a prominent calcium-independent SLAC1 phosphorylation activity similar to OST1. It was shown that CPK23 is active at resting cytosolic Ca2+ concentrations32, but cpk23 mutant showed no stoma phenotype, which was discussed as the functional redundancy with other CPKs. Further, it was suggested in the experimental literature that CPK23 may act as a negative regulator of stomatal closure in response to drought that activates ABA signalling in plant guard cells as cpk23 mutant displayed greatly reduced stomatal apertures36, while overexpression of CPK23 increased stomatal apertures. Moreover, another study29 showed that CPK6 and OST1 kinases strongly and independently activate SLAC1 in oocytes but another study37 showed that loss of function of CPK3 and CPK6 (double mutants) substantially reduced SLAC1 activity and ABA induced stomatal closure in plants. Another recent study done on plants showed that quadruple mutants of cpk3/6/5/11 did not strongly impair ABA induced stomatal closure38 indicating the existence of several regulatory mechanisms for SLAC1. These experimental findings are not consistent; hence, further analyses are required to understand biological mechanisms behind the role of CPKs. Sometimes, an adequate amount of OST1 may not be released into the cytoplasm at low ABA levels, and hence help of CPK may be needed for full activation of SLAC1 to close the stomata. At higher levels of ABA where there may not be such limitation of OST1 in the guard cell cytoplasm, OST1 may fully activate SLAC1 where CPK can further accelerate the activity of SLAC1 by stimulating peak activity or inhibiting slow inactivation39. Exploration of structural mechanisms between SLAC1 and two kinases, OST1 and CPKs, may reveal more evidence to narrow down the ambiguity between the two hypotheses. To date according to in planta studies, S59 and S120 phosphorylation sites in SLAC1 are required for intact ABA-induced stomatal closing but the molecular mechanism that translates the phosphorylation signal into the opening of the pore is still not understood. All these point to the possibility that SLAC1 may be regulated by at least two mechanisms in ABA signalling network, a ‘shortcut pathway’ activated by OST1 and Ca2+ induced second ‘loop pathway’38 activated by CPKs. Therefore, we assume Ca2+ elevation independent OST1 activation of SLAC1 in this study (This issue can be completely resolved only when definitive evidence is found in future). Background level Ca2+ was considered as the initial level of Ca2+ in the model because Ca2+ is one of the most ubiquitous molecules available at a low micro-molar level in plant cell cytoplasm19 regardless of active ABA signalling, hence requirement of background level Ca2+ for any communication was not considered in the model.A recent study2+ elevation did not influence the probability of closure  have been revealed since the 2006 ABA model and our model has incorporated them. This advancement of network topology improved the understanding of network functioning while preserving the core behaviour observed in the 2006 ABA model. This was because with the new network topology we were able to properly define some of the crucial regulators of the system and better define fine dynamics of the system . Therefore, the time taken by ABA signalling systems, one with Ca2+ signalling pathway intact and the other with no Ca2+ regulation, to achieve the final goal in response to a given set of initial conditions  was compared. Consistent with the previous model11, the results revealed that when the system has its Ca2+ regulation intact, the stomata close faster than in the Ca2+ knocked out system , which is 11% higher than the number of stomata closed in the Ca2+ independent system. In contrast, the highest percentage of stomata in the Ca2+ independent system closed within 12–13 min of getting the ABA signal. We repeated the same simulation with another two samples of 1,000 stomata and noted that, on average, 10–20% of the sample stomata were accelerated by Ca2+ elevations and the acceleration was by 0.5–5 min. Further, we observed that the majority of Ca2+ accelerated stomata (>25%) closed just 0.5 min faster and very few (<1%) showed 5 min accelerations. This indicates that the gain in acceleration is mostly around the lower end of the time scale (≈0.5 min). The model’s ability to shed light on these patterns of acceleration is another advantage of using real timing. From the biological perspective, even the slightest acceleration in stomatal responsiveness may still provide significant benefits to guard cells under actual stress conditions41. This is because as ABA induced stomatal closure is fast acting, even a slight acceleration can evade the negative control of elements that are at play to inhibit closure. Comparison of the degree of acceleration of the response time with the Mann-Whitney U test indicated that the time differences between the two systems are statistically significant with 0.02 probability of significance   and reversing the action of some Actin binding proteins to support stomatal closure. Since SLAC1 regulation and actin rearrangement are essential to close stomata, these findings signify the role of Ca2+ during the first phase of stomatal closure. These results conclude that ABA signal transduction can achieve improved rapidity in stomatal responsiveness with the elevated Ca2+ levels in the system indicating that Ca2+ elevation has a role to play during stomatal closure in addition to its potential role in closure maintenance.Further, we identified that this acceleration in closure is primarily due to the time taken to stabilize the activity of SLAC1 and actin rearrangement. The activation time for both SLAC1 and actin rearrangement is significantly lower in the Cae) Table . This is2+ elevation was shown to be potentially non-essential in the first phase of ABA signalling but shown to accelerate closure, we checked for any other secondary roles of Ca2+. The previous model12 reported that perturbations of Ca2+ (or Ca2+ influxes), when combined with disruptions in PLD (Phospholipase D), PA, GPA1 (G protein α subunit1), or pH, lead to stomatal insensitivity to ABA response. Their results signified that Ca2+ elevation is required for the system to close stomata when pH, K+ efflux or the S1P (Sphingosine-1-phosphate) –PA pathway are perturbed but how Ca2+ safeguards the signalling flow of these pathways still remains to be explored.As the role of Ca2+ elevation in ABA signalling network resists these reported perturbations and if there are any other such important perturbations. The importance of Ca2+ in resisting perturbation to important functional events of the system was evaluated by comparing the system performance in the wild type with the Ca2+ knocked out system when functional links or elements of the important functional sets , cytoskeleton rearrangement (Actin), ROS signalling (ROS) etc.,) are perturbed. However, results showed that Ca2+ plays an important supporting role in the absence of upstream regulatory elements or functional links of these hub elements by acting as a balancing loop to continue the downstream signalling flows. It was noted that some of the findings of the previous model12 are conserved in our model results; for example, perturbed lipid pathway  or plant pH together with Ca2+ produced drastically impaired stomatal closure  but undisturbed Ca2+ elevation becomes beneficial to the system to withstand such perturbations 47, which is an essential component for SLAC1 activation. Moreover, ROS regulates actin rearrangement via facilitating disassembly of actin filaments by weakening the inter-monomer bonds of the filaments48. Guard cell Ca2+ signalling is also supported by ROS via initiating Ca2+ influxes to the cytosol by adjusting the gating properties of the plasma membrane Ica channels49 and further facilitating NO production50 to pump Ca2+ from the internal organelles. Lipid signalling, especially Sphingolipid signalling, is very important to generate phosphatidic acid (PA), which mediates all the functional sets of the ABA signalling through the production of ROS51, inhibition of PP2C52 (Protein phosphatase type 2C) and inhibition of AHA153. We observed that perturbed lipid pathway  altered the production of ROS and thereby altered osmoregulation and structural rearrangements resulting in defective stomatal behaviour in response to ABA. Of the two perturbed systems, the one with intact Ca2+ repeatedly attempts to reach stomatal closure by evoking Ca2+ dependent pathways  on both graphs in Fig. 2+ is available guard cells achieve a better steady level closure. Near normal activity of SLAC1 was observed in ~50% of the sample stomata with undisturbed Ca2+ signalling pathway. The model showed that this is due to Ca2+ that attempts to restore the ROS production and thereby activate MAPK (mitogen activated protein kinase) which is an essential regulator of SLAC1 by inducing respiratory burst oxidase, RbOH, via CPKs. The model further reveals that restoration of ROS production by Ca2+ also stabilises structural rearrangements. This has support in the literature such that ROS supports structural rearrangement of guard cells via regulation of actin-related protein-2/3 (ARP2/3)54 and PI3P (Phosphatidylinositol 3-phosphate) mediated actin depolymerization by weakening the inter-monomer bonds of actin filaments48. Thus our model was able to elucidate how Ca2+ becomes required for engendering stomatal closure when upstream regulators of ROS production are perturbed by revealing the importance of ROS production through Ca2+ dependent pathway consistent with experimental literature55. In addition, literature supports that Ca2+ itself also acts as a mediator for guard cell cytoskeleton rearrangement on concentration basis, such that at low Ca2+, actin filaments are bundled by actin crosslinking proteins, whereas elevated Ca2+ levels modulate capping and depolymerization56, facilitating the disassembly of actin, and our model has captured these regulatory mechanisms.Our model further identified that this stomatal impairment is because lipid pathway and pH transmit the drought signal to keep the guard cell ROS production stable see Fig. . ROS is ays Fig.  to stabi2+ promotes stomatal closure by facilitating osmoregulation through regulation of the vacuolar malate transport channels . Moreover, the model output displayed a reduction in SLAC1 activity when both QUAC and Ca2+ were perturbed but restored SLAC1 channel when Ca2+ was re-established in the system indicating the interconnectedness of signalling events57  type malate efflux channel, an essential element in osmoregulation)  as shown in red colour panels in Fig. 2+, closure tends to show oscillatory behaviour but Ca2+ elevations tend to stabilize stomatal closure by acting as a balancing loop that minimizes the fluctuations and, thereby reduce the potential damage to guard cells , ARP2/3, ABPs (Actin binding proteins) and ATRAC1 (Rho-related GTPases), as in Fig. 2+ regulatory systems and their links to other important functional sets of ABA signalling were refined. We showed that the Asynchronous Boolean ABA model that was updated using real timing allowed us to produce system outputs close to biology . Thus, the findings from our model are biologically validated and, therefore, insights from the model are useful. The model elucidated the role of Ca2+ and related mechanisms in greater clarity and detail than before. Specifically, it predicts that Ca2+ elevation is essentially not needed in rapid stomatal closure as stomata can close independently of elevating Ca2+. However, the system benefits in various ways from having Ca2+ in the system as a balancing loop to achieve faster and steady level closure. The previous model also predicted that Ca2+ can accelerate stomatal closure. Our model was able to provide the reason for it which is that Ca2+ accelerates stomatal closure through enhancing the activity of SLAC1 and actin rearrangement. Another new finding is that the model results identified SLAC1 as the most sensitive to Ca2+ regulations, much more so compared to GORK, and it was supported by experimental literature33.In this study, we assembled an extended ABA network with the most current knowledge available at the time that incorporated a number of new important regulatory nodes and links. In particular, Ca2+ in enhancing closure resilience, similar to the previous model. With our model, we could go further to answer the question of how Ca2+ elevation enhances resilience  of the system. According to the results, the cross-talk between Ca2+ and ROS, also supported by literature58, strengthens SLAC1 activity via direct regulation of CPKs by Ca2+ and indirectly via ROS regulation to induce MAPK, an essential regulator of SLAC1. In addition to these molecules, GHR1 (Guard Cell Hydrogen Peroxide-Resistant1) is also known as an essential regulator to activate SLAC1. The most recent literature observes that ROS and MAPK are involved in regulating SLAC1 through GHR1 in CO2 induced signalling59 but is not confirmed in ABA signalling. It was further observed in the oocytes expression system that GHR1 physically interacted with, phosphorylated, and activated SLAC160. In the absence of confirmed mechanism of these molecules in guard cell ABA signalling, we assumed that ROS is an essential regulator for MAPK and MAPK is essentially required for activating SLAC1. As we have used timing from MAPK to SLAC1, ignoring putative nodes in between may not make a difference to the model output unless a novel regulatory mechanism for GHR1 is revealed. However, GHR1 also can be added to the model for obtaining more precise output once more information is available on this regulation. The cross-talk between Ca2+ and ROS regulating pathway further supports guard cell structural rearrangement by providing balancing loop regulations to Actin regulating proteins such as ARP23.Further, our model predicted the role of Ca2+ dynamics: It revealed biologically/experimentally consistent timing as reported in the literature for activation of Ca2+ during stomatal closure. It produced the parameters of Ca2+ oscillations (amplitude and duration)  closer to reality as well as the timing of occurrence of related events in the Ca2+ system. It helped quantify the level of acceleration of closure due to Ca2+and show that Ca2+ induced acceleration is statistically significant. It also allowed us to show that Ca2+ oscillations are sustained without decay in our model due to longer positive feedback loops. Previous model proposed this as a requirement for sustained oscillations and our model was able to confirm it with evidence. Further, timing helped explain the reason for a number of observations from the previous model and our model . Timing also helped us reveal the statistically significant reduction in activation time for both SLAC1 and actin rearrangement in the Ca2+ dependent system compared to Ca2+ independent system.Updating the model using real timing provided useful insights into Ca2+ independent signalling pathway to regulate stomata if the OST1/SLAC1 pathway works independently of Ca2+ to regulate stomata. In the absence of evidence for OST1 and SLAC1 relationship directly from planta data, the Ca2+ independence found for plant OST1 and SLAC1 in oocytes was used in this study. Currently, the evidence for independence as well as somewhat inconsistent evidence against it exist. Our results in terms of timing of ABA events and Ca2+ dynamics were shown to correspond well with biology. However, future experiments may confirm the validity of this assumption. Further, the results indicate the dependence of Ca2+ dependent pathway on Ca2+ independent signalling pathway as the former could not achieve stable stomatal closure as evident in the latter case. This is because CPK, the main protein that decodes the Ca2+ signal, shows oscillatory behaviour that in turn leads to oscillations in stomatal closure. However, the behaviour of Ca2+ governed CPKs should be biologically validated. Additionally, our model used evidence in literature that SCAB1 is essential for actin rearrangement and it is regulated by ABA. Results indicate that if future experiments reveal a connection between SCAB1 and Ca2+ and/or CPKS, possibility exists for Ca2+ to take the role of ABA upon its discontinuation.Our model supports the hypothesis of existence of a Ca2+ sensitivity priming hypothesis at physiological resting Ca2+ levels as the mechanisms to support this hypothesis need further exploration. There are few evidences for existence of Ca2+ sensitivity priming in plants 63 but future research needs to show that Ca2+ independent signalling in guard cells is truly Ca2+ dependent to support this hypothesis. In our study, basal Ca2+ did not activate CPKs because Ca2 + → CPK interaction was defined in the model such that above basal Ca2+ concentration is required to activate CPKs based on the structural properties of CPKs. We plan to explore Ca2+ sensitivity priming issue with our model at the next stage of research as considerable activities of some CPKs at basal Ca2+ level have been reported64. Whether CPKs are essential regulators in activation of SLAC1 in response to ABA in guard cells requires further clarification. However, we can hypothesise that if OST1 is a critically limiting component in ABA signalling65 as it has multiple essential phosphorylating targets, SLAC1, QUAC and RbOH, then some mutual equilibrium between OST1 and CPKs may reinforce regulation of SLAC1 to its full potential.The model does not support or oppose the recently proposed Cafast-acting protein signalling system that does not involve gene expression. Our model outcomes provide avenues for future experimental investigations to further consolidate the understanding gained here. In this paper, our focus was on elucidating the role of Ca2+ in stomatal closure. Soon, we also intend to report on model outcomes for the whole ABA system response from the perspective of, and highlighting, all its functional sub-systems, including Ca2+ sub-system, to elucidate how the system regulates them concurrently in a well-coordinated and timely manner to achieve stomatal closure. In particular, in future, the new knowledge of Ca2+ and our model could potentially help develop drought resistant plants that survive the extremes of climate change.Considering the emerging understanding that the timing is more crucial than the exact level of proteins, we believe that the system dynamics revealed by our model is a closer qualitative approximation of the real system based on our current understanding of ABA signalling. Further, having greater details of the continuously changing levels of proteins  may not provide much additional insight into this The extended network shown in Supplementary Fig. We defined a Boolean network (Supplementary text 1) in such a way that the logic functions define the state of system variables  in qualitative form denoted by 0 (below a threshold) or 1 (above it). We used logical operators “AND”, “OR” and “NOT” in Boolean functions to represent the influence of a node (variable) on another considering biological interactions and their mode of actions Table . OperatoBi is the Boolean function associated with node i and τji are the corresponding edge delays for a regulator node j of node i.We incorporated network node edge delays (time delays) into an asynchronous updating scheme in order to provide the network nodes with realistic biological asynchrony. Here we assumed that proteins get activated according to the time required for the activation of their regulators. Therefore, in this analysis, we updated a network node at time ‘t’ based on the status and the processing time of each of its immediate regulators Eq. . The nodτ) of a reaction. For clarity, for a simple example of a three node network where A → B ← C, edge delays are defined in such a way that if A induces half activation of B within τA and C half activates B within τC, τA and τC are considered edge delays associated with node B.Through an extensive literature survey, we collected research findings on time delays of ABA signalling network nodes defining edge delay as the biological half activation time (τ1) and B → C (τ3) are known, then the time scale for A → B (τ2) is calculated as the difference τ1-τ3.In situations where no direct information is available (roughly 30% of interactions), we collected data from similar interactions occurring in other signalling systems in plants, animals or humans. When edge to edge timing is not available for an intermediate step, we calculated time scales by decomposing a (larger) available time scale into a set of smaller time scales using a simple algebraic formula such that, for instance, if node A activates node C via node B (A → B → C), where the time scales for A → C  were in physiologically acceptable levels for plants, despite system, varietal, and kingdom level differences69.As a consequence of the unavailability of a single data set for the timescales of the ABA system, relative timings of the network edges were extracted from various sources; hence, the heterogeneity in information pool may introduce some noise to the model results. Therefore, the data set was carefully scrutinised to minimize the noise as this was considered the best option for the model. We observed a number of favourable justifications for pooling information from different sources in that the time delays in plant, human and animal systems were comparable in many reactions and time delays were consistent between ‘We collected experimental results from literature for all network reactions and calculated the mean activity time for each, which correspond to the biological timeframe of ABA induced stomatal closure. According to the data collected, time delays in the current system ranges from milliseconds to minutes (Supplementary Table Interactions for which there was no information available in the literature (about 10%), we made realistic heuristic assumptions considering the most relevant factors/knowledge. In some situations, we generalized a reaction such that if a particular protein is phosphorylated independently by a number of kinases, we assumed that all such reactions happen on a similar timescale . Similarly, if one protein dephosphorylates several kinases, we assumed that all such reactions happen on a similar timescale ). In some other cases such as the catalytic effect of an enzyme changing the concentration of an end product through an intermediary (OST1 → RbOH → ROS) where the time for each step is not known but the total time is known, the second step was assumed rapid. Finally, for non-biological nodes like ‘CLOSURE’, the smallest timescale was assumed for all its regulators.τB and τC are the edge delays from B to A and C to A, respectively, We defined operators for the updating scheme considering the logic gates of corresponding nodes , the updating operators are defined as in Eq. a priori. The probability of success  was obtained from Eq. et al.11.jth simulation and N is the total number of simulations.With Boolean functions and associated time delays, the network is simulated for 100 discrete time points with a large number of random initial states  to study the most frequently visited states/attractors of the network. The state of input node ABA was always kept at on (1) state and the target node (CLOSURE) set at the off (0) state for all initial conditions. As the stomata is considered in the open stage, the initial conditions of nodes, which satisfy the stomatal opening were considered active  = 1, AHA1 = 1, DEPOLAR (Plasma membrane depolarisation) = 0, PYR = 0 and PP2C = 1) when the simulation begins. Random initial states were assumed for all other nodes (>80%) where initial states were not known The significance of a specific node on the global behaviour of the network was evaluated by keeping its state below the threshold level (0) over the simulation to reproduce biological knockouts and/or pharmacological inactivation. Further, overexpression of a particular node was mimicked by constantly expressing its state above the threshold level (1) throughout the simulation period.2+ to the system behaviour was studied by setting the state of Ca2+ node at inactive state (0) over the simulation. In order to see if having Ca2+ in the system accelerates closure, we compared the timing of stomatal closure for WT and Ca2+ knocked out systems. Comparison was done between time taken to close stomata by the two systems in response to a given set of initial conditions. Initial conditions used in the simulation were identical for both systems. We used 10,000 random initial conditions to simulate the dynamics of both systems. Time differences between the two systems for similar initial conditions were compared using nonparametric version of paired t-test, Mann Whitney U test, as the data set did not follow a normal distribution. Whether Ca2+ plays a role in improving system robustness was tested by perturbing crucial elements in the functional sets in the WT and Ca2+ knocked out systems and comparing the difference in the stability of stomatal closure.The significance of CaSupplementary Information


Within insulin-producing β-cells, increases in cytosolic Ca2+ directly stimulate insulin vesicle exocytosis, but also initiate multiple signaling pathways. Mediated through activation of downstream kinases and transcription factors, Ca2+-regulated signaling pathways leverage substantial influence on a number of critical cellular processes within the β-cell. Additionally, there is evidence that prolonged activation of these same pathways is detrimental to β-cell health and may contribute to Type 2 Diabetes pathogenesis.The divalent cation Calcium phosphorylation events and activation of downstream transcription factors. The short-term stimulation of these Ca2+ signaling pathways promotes numerous cellular processes critical to β-cell function, including increased viability, replication, and insulin production and secretion. Conversely, chronic stimulation of Ca2+ signaling pathways increases β-cell ER stress and results in the loss of β-cell differentiation status. Together, decades of study demonstrate that Ca2+ movement is tightly regulated within the β-cell, which is at least partially due to its dual roles as a potent signaling molecule.Acute increases in intracellular Ca  These processes are triggered within microseconds of Ca2+ influx into the cytosol 2+i that persist for seconds to minutes produce long-term responses, dependent on the activation of downstream signaling pathways 2+ signaling cascade contributes to the dysfunction of multiple tissues and cell types in metabolic disorders Elevated cytosolic Ca2+i causes insulin granule exocytosis, but Ca2+-mediated signaling pathways also have critical roles in promoting the function, survival, and proliferation of these cells. This review aims to highlight sources of Ca2+i, important mediators of β-cell Ca2+ signaling and their relevance to β-cell biology and type 2 diabetes (T2D).Within insulin-producing β-cells, increased Ca22+i that effectively stimulates insulin exocytosis from the β-cell must be closely regulated. This tight control requires the cooperation between multiple Ca2+ exchangers, pumps, and channels β-cells regulate the systemic response to hyperglycemia through the production and secretion of the hormone insulin. Given the detrimental effects of either impaired or elevated insulin release, the increase in Ca2+ through L-type voltage-gated Ca2+ channels (L-VGCCs). Mediated via glucose metabolism and ATP production, shifts in the ratio of ATP to ADP (ATP:ADP) within the β-cell result in the closure of ATP-sensitive potassium (KATP) channels and membrane depolarization. In human β-cells, L-VGCCs are activated at a membrane potential of −40 mV and, in concert with T-type and P/Q-type Ca2+ channels, allow Ca2+ influx to elicit insulin exocytosis 2+ flux across the β-cell plasma membrane is further regulated by a number of metabolites and nutrients including free fatty acid signaling and cAMP 2+ channels 2+ influx.In the postprandial state, glucose elicits the influx of Ca2+, there are multiple membrane-bound organelles that regulate Ca2+i levels, including the nucleus, endoplasmic reticulum (ER), mitochondria, Golgi, as well as vesicles and granules 2+ stores are distinguished based on their sensitivity to inositol-1,4,5-trisphosphate (IP3), nicotinic acid adenine dinucleotide phosphate (NAADP), or ryanodine , which is expressed on the ER membrane 3 is generated downstream of certain Gαq-associated G protein coupled receptors, such as the free fatty acid receptor GPR40 (FFAR1), which signals through phospholipase C (PLC) 3. Alternatively, PLC is also activated by an increase in Ca2+i; suggesting other sources of Ca2+  can trigger release of Ca2+ from IP3R-responsive stores Ca2+i is the NAADP-responsive pool. Within β-cells, NAADP acts as a second messenger of glucose metabolism, as elevated glucose exposure rapidly increases β-cell NAADP content 2+ release from acidic vesicles such as lysosomes and insulin granules 2+i, as their inhibition is sufficient to impair glucose-stimulated insulin secretion A second source of Ca2+i is the ryanodine-sensitive pool + by ARC enzymes, including CD38 2+-induced Ca2+ release (CICR) 2+ store expresses RyRs. With ER-localized RyRs, CICR is thought to increase Ca2+i in close proximity to mitochondria and maintain high rates of ATP generation. Similarly, RyRs expressed on the insulin granule increase Ca2+i in the immediate proximity of the insulin granule and facilitate Ca2+-dependent vesicle docking and fusion The third source for Ca2+-sensitive stores, mitochondria are additional β-cell organelles in which Ca2+ handling is tightly regulated and critical for function 2+ is exported from mitochondria via the Na+/Ca2+ exchanger (NCLX) 2+ influx into the mitochondrial matrix is achieved through voltage-dependent anion channels (VDACs) in the outer mitochondrial membrane, and the mitochondrial Ca2+ uniporter (MCU) complex in the inner mitochondrial membrane 2+ uptake 1 (MICU1) is critical for mitochondrial function and β-cell function, as knockdown of MICU1 in INS1 cells reduces Ca2+ influx into mitochondria, resulting in reduced glucose-stimulated mitochondrial respiration, ATP production, and insulin secretion 2+ entry from the cytosol is limited by the low affinity of the MCU, but microdomains between the ER and mitochondria , tethered through GRP75 and mitofusin 1 and 2, facilitate the rapid transport of large quantities of Ca2+ from the ER into mitochondria following IP3R- or RyR2-mediated ER Ca2+ release 2+ influx into the mitochondria during periods of high metabolic demands ensures adequate ATP production to maintain insulin secretion by increasing the availability of metabolic substrates and stimulating the TCA cycle (possibly through activation of 2-oxoglutarate dehydrogenase and isocitrate dehydrogenase) 2+ levels.Beside the regulation of these Ca2+i levels through the regulation of extracellular Ca2+ influx and the movement of Ca2+ within intracellular depots. The degree of this complexity is illustrated through Ca2+ microdomains. Basal levels of free intracellular Ca2+ are approximately 100 nM, 20,000× lower than free extracellular Ca2+. Following stimulation, whole cell Ca2+i increases to 300–1000 nM, but more responsive Ca2+ microdomains exist within multiple subcellular locales including dense core vesicles, ER, mitochondria, subplasmalemma, and within the nucleus β-cells maintain tight control of Ca2+ is required for activation of cAMP response element binding (CREB) 2+ microdomains surrounding dense core vesicles have been postulated to amplify insulin secretion by increasing Ca2+ concentrations in close proximity to Ca2+-dependent synaptic proteins 2+ microdomains formed within the mitochondria following high glucose exposure are required for mitochondrial function and second phase insulin secretion Each of these microdomains have functional consequences. The increase in nuclear Ca2+ handling is highly complex, requiring multiple receptors and channels on multiple organelles and the plasma membrane. The potency of Ca2+ as a signaling molecule is a major reason for this degree of intricacy.The regulation of Ca32+i initiates multiple signaling cascades by binding to and activating the Ca2+ sensor protein Calmodulin (CaM). CaM then undergoes a conformational change, allowing it to activate numerous downstream targets 2+i conditions Once elevated, Ca2+/Calmodulin-dependent protein kinases (CaMK) are one class of proteins activated by Ca2+-bound CaM. Of the CaMK isoforms 2+ conditions, inactive CREB is bound to consensus sites (TGACGTCA) 2+i result in CREB activation through a CaMK-dependent pathway 2+i-mediated phosphorylation of CREB, Ca2+ signaling pathways also increase CREB activity via CRTC2. Activation of the phosphatase Calcineurin (CaN) results in the dephosphorylation of cytoplasmic CRTC2, which subsequently dissociates from cytoplasmic 14-3-3 chaperone proteins and translocates to the nucleus, where it increases CREB transcriptional activity The CaIndependent of the CaMK/CREB pathway, CaM also activates a separate signaling cascade through CaN. CaN has many target proteins, including nuclear factor of activated T cells (NFAT) 2+i in β-cells activates other proteins and signaling cascades, including the MAP kinase pathway. This is mediated through the activation of Ras-GEF and B-Raf via CaM 2+i in β-cells. CaMKII mediates the activation of NF-κB activation in β-cells through the phosphorylation of IκBα 2+i of each of these pathways are not well defined in β-cells. While computational modeling predicts that increasing frequency of Ca2+i oscillations preferentially activates CaMKII over CaN In addition to CaMK and CaN pathways, increased Ca2+i suggest that Ca2+ is a central mediator of many different cellular processes within the β-cell. Indeed, the study of the mediators and effectors of these Ca2+ signaling pathways demonstrates their importance in maintaining β-cell function and glucose homeostasis.The disparate pathways active by elevated Ca42+ is critical for this process, as blocking L-VGCCs with verapamil ameliorates glucose-mediated insulin transcription 2+-mediated promotion of insulin transcription can be separated into NFAT- and CaMK-dependent pathways. The rat insulin 1 promoter contains multiple NFAT binding sites 2+ signaling pathways; therefore, conclusions derived from cell lines should be verified in primary cells. Furthermore, inhibiting NFAT with the CaN inhibitor tacrolimus (FK-506) abrogates the glucose-mediated increase in insulin promoter activity in INS-1 cells Ins1 and Ins2 gene expression in vivoDuring periods of elevated metabolic demand, β-cells must increase the production of insulin to ensure adequate insulin stores are maintained. As such, high glucose exposure increases insulin production in rat islets 2+ in CaMKIV- and SRF-dependent manners, respectively, and overexpression of either factor is sufficient to increase insulin promoter activity 2+, acting as a stimulus for insulin secretion and also a signal to increase insulin synthesis, ensures adequate insulin levels during prolonged stimulation. In addition to the transcriptional regulation of insulin by members of Ca2+ signaling pathways, elevated glucose also increases rates of insulin mRNA translation 2+ signaling pathway members in these processes is unknown.Besides NFAT, CaMKIV is also required to induce insulin expression, as shown by transfection of INS-1 cells with a kinase-dead CaMKIV, which blocks glucose-mediated elevations in insulin promoter activity 2+i are required for insulin granule fusion to the plasma membrane, activation of Ca2+ signaling pathways also promotes insulin secretion through CaMK- and CaN-dependent pathways. The importance of Ca2+ signaling pathways in promoting insulin secretion is observed in mouse models wherein diminished activity or expression of CaMKII in vitro and in vivo models all support a critical role for members of Ca2+ signaling pathways in the promotion of insulin secretion.While increases in Ca2+ signaling promotes insulin secretion is through the formation β-cell “metabolic memory”, wherein repeated exposure to elevated glucose primes β-cells to significantly increase insulin secretion during an ensuing high glucose exposure One mechanism through which Ca2+ signaling may also promote insulin secretion by elevating mitochondrial activity through a process termed “Ca2+-metabolic coupling”. Periods of elevated insulin secretion require increased mitochondrial activity to replenish the ATP stores that sustain ATP-mediated membrane depolarization and insulin release. Influx of Ca2+ and downstream activation of CaMKs is required for this prolonged elevation in mitochondrial function, as inhibiting L-VGCCs or CaMKs blocks increased oxygen consumption rate  2+i with mitochondrial function Ca2+-mediated insulin vesicle fusion, activation of CaN/NFAT and CaMK also promote insulin secretion by increasing mitochondrial respiration and priming the β-cell under repeated high glucose exposures.These studies establish that, in addition to Ca5in vitroin vivo studies 2+ signaling is critical for this process. Pharmacologic stimulation of glucokinase also increases β-cell replication 2+ influx, as opposed to glucose metabolism alone, is necessary. Furthermore, increasing Ca2+i with the L-VGCC agonist, BayK8644, induces rat β-cell proliferation 2+ signaling pathways in promoting β-cell proliferation.Increased rates of β-cell proliferation are one adaptive mechanism β-cells employ to compensate for elevated metabolic demand and ensure euglycemia is maintained. Both 2+i in β-cells. Blocking CaMK activity with KN62 abrogates the glucose-mediated increase in β-cell proliferation Irs2 and Nr4a1 promote β-cell proliferation Irs2 and Nr4a1 pathway is one mechanism by which elevations in Ca2+i promote β-cell replication.Both CaMK- and NFAT-dependent mechanisms mediate the mitogenic effects of elevated CaNFATC1, NFATC2, and NFATC4 than islets from adults (20 years or older) in vivoNFATC1, NFATC3, and NFATC4 expression. Inhibition of CaN with FK-506 abrogated exendin-4-mediated increases in NFAT gene expression level and β-cell proliferation rates Irs2Ccdn1, and Cdk4NFAT proteins also promote β-cell replication. Islets from juveniles (age 0.5 to nine years old) have higher proliferation rates associated with higher expression of 2+ signaling pathways on insulin production, elevated β-cell proliferation rates during periods of increased systemic insulin demand allow for appropriate β-cell compensation and ensure appropriate β-cell functional capacity to maintain euglycemia.Similar to the positive effect of Ca62+ signaling pathways also promote β-cell viability and survival. MIN6 cells incubated for 24 h in high glucose (25 mM) have significantly reduced rates of apoptosis compared to MIN6 incubated in low glucose (5 mM) concentrations 2+ influx with nifedipine 2+.Cain vitroInhibition of CaN with either FK-506 or cyclosporine A induces β-cell apoptosis in human islets in vivo studies show that transgenic expression of a dominant-negative CREB (A-CREB) in β-cells increases apoptosis and results in diabetes in mice in vitro studies demonstrate knockdown of CREB in INS-1 cells increased levels of cleaved caspase-3 Irs2 and Npas4, which both protect β-cells from stress and cell death In addition to the role of NFAT proteins, the CaMKIV/CREB pathway also promotes β-cell viability. MIN6 cells incubated in 12.5 mM glucose have significantly reduced caspase-3 activity compared to MIN6 incubated in 2.5 mM glucose 2+i to promote β-cell viability, decreased Ca2+i also adversely impacts β-cell survival by impairing ER and mitochondrial Ca2+ handling. Depletion of ER Ca2+ results in ER stress and β-cell apoptosis 2+i, such as low glucose exposure, ER Ca2+ depletion occurs due to inactivation of the sarcoplasmic endoplasmic reticulum Ca2+ ATPase (SERCA) and the ensuing lack of ER Ca2+ uptake 2+i is also intricately connected to mitochondrial function, as the activity of several mitochondrial enzymes depends on Ca2+2+ uptake follows depolarization-dependent increases in Ca2+i, and ATP production increases as a consequence 2+i are predicted to decrease mitochondrial activity, ATP production and SERCA action; and in so doing promote ER Ca2+ depletion, ER stress and β-cell death.In addition to the ability of elevated Ca72+ signaling pathways have critical roles in regulating β-cell function, proliferation and viability; all processes that fail during the development of T2D. Despite this importance, only few members of Ca2+ signaling pathways have appeared as susceptibility loci in T2D GWAS studies CDC123/CAMK1D locus CAMKK2 variants NFATC2 as a regulatory factor for 40% of genes identified through GWAS KLF11, HHEX, and PROX12+ signaling pathways and T2D requires further examination, research using animal models and clinical data support a role for impaired Ca2+ signaling in β-cell failure during T2D pathogenesis.As outlined above, Ca2+ influx, and insulin secretion to maintain euglycemia. Short-term stimulation of Ca2+ signaling pathways yields positive effects for the β-cell . In contrast, chronic stimulation of these pathways has deleterious effects. This is observed in multiple rodent models in which members of Ca2+ signaling pathways are overexpressed. For instance, overexpression of a constitutively active CaN increases β-cell apoptosis, decreases proliferation, and results in glucose intolerance 2+ signaling pathways impairs β-cell function, which is supported by human studies in which individuals with T2D are treated with diazoxide to inhibit β-cell depolarization. After a multi-day treatment period, insulin secretion is improved 2+ flux may also explain why sulphonylureas initially improve, but ultimately worsen, glycemic control in individuals with T2D The prediabetic milieu, characterized by increased glucose and fatty acids levels, results in increased β-cell depolarization, Ca2+i may drive β-cell dysfunction and failure by exacerbating ER stress and β-cell differentiation. β-cell ER stress is sufficient to cause diabetes in mice 2+ signaling pathways 2+ influx with nifedipine Chronically elevated Ca2+ signaling also results in loss of β-cell maturation. Models in which β-cells are exposed to chronically elevated glucose levels and increased Ca2+i result in the loss of β-cell maturation; such as the db/db or Akita mouse, a diphtheria toxin-mediated β-cell ablation model, insulin receptor antagonism db/db mouse, by crossing it to a β-cell specific constitutively active Kir6.2 mutant, significantly reduces rates of β-cell transdifferentiation into gastrin-expressing cells compared to db/db controls, despite no improvement in glucose handling in vitro transdifferentiation model in which mouse islets are cultured at either 5 mM or 25 mM glucose. After 2 days in culture, islets exposed to high glucose have a significant increase in gastrin expression, which could be abrogated by co-culture with either diazoxide, nifedipine or FK-506 2+ influx and CaN activity are also required for this process. Importantly, islets from individuals with T2D have significantly increased numbers of gastrin-expressing cells; although it remains unknown whether aberrant Ca2+ signaling is the cause In addition to exacerbation of ER stress, chronically active Ca2+ signaling in driving β-cell transdifferentiation is further supported by data from the β-cell Abcc8 knockout mouse. Deletion of Abcc8, a subunit of the KATP channel, from β-cells increases intracellular Ca2+ most notably under low glucose conditions, but also under high glucose exposure Aldh1a3 is significantly increased in the Abcc8 null mouse and can be largely normalized by blocking Ca2+ influx with verapamil Abcc8 null model decouples hyperglycemia from increased Ca2+ influx and elegantly demonstrates that chronically active Ca2+ signaling pathways are sufficient to promote β-cell dedifferentiation.The role of Ca2+ signaling pathways in driving β-cell dedifferentiation and transdifferentiation and support a model in which chronic activation of Ca2+ signaling pathways results in increased stress and a loss of β-cell maturation status that contributes to β-cell failure in T2D. Less certain, however, is the role of altered Ca2+ handling in the development of type 1 diabetes (T1D). In vitro models of cytokine treatment show impaired β-cell Ca2+ handling following exposure to proinflammatory cytokines, including reduced oscillatory Ca2+ fluctuations 2+ influx 2+-regulated cell death in T1D.These studies define a clear role for Ca82+ influx, which activates multiple downstream signaling pathways. Stimulation of these pathways promotes insulin production and secretion, proliferation, and viability. The importance of Ca2+ signaling in β-cells is evidenced by the β-cell dysfunction and impairment in systemic glucose homeostasis that results from inhibiting the activity of various members of the Ca2+ signaling cascade, including CREB 2+i and the downstream pathways it regulates in β-cells.In the postprandial state, β-cells undergo waves of depolarization and Ca2+ handling in mouse models 2+ signaling in the β-cell. These include a further characterization of how altered Ca2+ signaling impacts β-cell transcriptomics, ER and mitochondrial function, and defining the nature of the altered Ca2+ handling by the β-cell under pathologic conditions, particularly by important Ca2+ stores such as ER and mitochondria. Finally, it will be important to determine if and how Ca2+ signaling pathways are impaired in β-cells from individuals with T2D and whether these pathways can be therapeutically targeted.While current studies have observed disrupted β-cell Ca


However, the sub-organellar Ca2+ kinetics remain elusive. In the present work we identified novel site-specific targeting sequences for the intermembrane space (IMS) and the cristae lumen (CL). We used these novel targeting peptides to develop green- and red- Ca2+ biosensors targeted to the IMS and to the CL. Based on their distinctive spectral properties, and comparable sensitivities these novel constructs were suitable to visualize Ca2+-levels in various (sub) compartments in a multi-chromatic manner. Functional studies that applied these new biosensors revealed that knockdown of MCU and EMRE yielded elevated Ca2+ levels inside the CL but not the IMS in response to IP3-generating agonists. Knockdown of VDAC1, however, strongly impeded the transfer of Ca2+ through the OMM while the cytosolic Ca2+ signal remained unchanged. The novel sub-mitochondrially targeted Ca2+ biosensors proved to be suitable for Ca2+ imaging with high spatial and temporal resolution in a multi-chromatic manner allowing simultaneous measurements. These informative biosensors will facilitate efforts to dissect the complex sub-mitochondrial Ca2+ signaling under (patho)physiological conditions.Mitochondrial Ca The molecular nature of the mitochondrial Ca2+ uniporter is a complex formation of several interacting proteins located in the IMM including the pore forming subunit mitochondrial Ca2+ uniporter (MCU) (2+ uptake 1 and 2 (MICU1 and MICU2)   to the IMS or the mitochondrial cristae. We identified specific targeting sequences for distinct sub-mitochondrial protein targeting and fused them with single FP-based various GeCO1-like Ca2+ indicators  compartments and enabled us to take a closer look in the kinetics of Ca2+ transfer from the ER to mitochondria.Embedded between the outer (OMM) and the inner mitochondrial membrane (IMM) the mitochondrial intermembrane space (IMS) including the cristae lumen (CL) represents the smallest mitochondrial sub-compartment. This specialized area is of upmost importance for the regulation of mitochondrial protein import, lipid homeostasis, energy metabolism and metal ion exchange to maintain cellular functions . While ter (MCU) , the esser (MCU)  and the d MICU2) . Recentl the IMS , which is (MAMs) . The forganelles . On the lles  composed of (in mM): 2 CaCl2 135 NaCl, 5 KCl, 1 MgCl2, 1 HEPES, 10 D-glucose, pH adjusted with NaOH to 7.4 and stimulated with either BHQ or histamine in either a Ca2+ free solution or a nominal Ca2+ free solution containing EGTA instead of 2 CaCl2.Chemicals and reagents for solutions and buffers were purchased from Carl Roth . Histamine, monensin, and BHQ were obtained from Sigma-Aldrich . Nigericin was from Tocris  and ionomycin from Abcam Biochemicals . For storage cells were maintained in a buffer (storage buffer) containing (in mM): 2 CaCl2/95% air). Cells were transfected in a serum-free medium using 3 μg/ml TransFastTM Transfection reagent  together with 1–2 μg of plasmid(s) and/or 100 nM of the respective siRNA(s) per ml 2 days before experiments.In this study we exclusively used a HeLa cell model that was cultivated at passage >50 on low fluorescence glass cover slips with a diameter of either 15 or 30 mm in DMEM  containing 10% FCS , penicillin (100 U/ml) and streptomycin (100 U/ml) in a humidified incubator . All siRNAs were purchased from Microsynth  and their  sequences were: ACACUAGGCACCGAGAUUA (siVDAC1) for silencing hVDAC1  was synthesized by General Biosystems Inc.,  and the vectors encoding for GEMGeCO1, CARGeCO1, and mt-CARGeCO1 were purchased from Addgene  and used as vector templates for the N-terminal insertion via g hVDAC1 ; GCCAGAGg hVDAC1  and thusg hVDAC1 .® qPCR Master Mix  together with gene-specific primers . Experiments were performed on a LightCycler 480 . Relative expression of specific genes was normalized to human GAPDH, as a housekeeping gene. Primer sequences were as follows: VDAC1 for: 5′-GGACTGAGTACGGCCTGACGTT-3′, VDAC1 rev: 5′-CAGTCCACGTGCAAGCTGATCT-3′, GAPDH .Total cellular RNA was isolated from HeLa cells transfected with either siRNA against VDAC1 or the scrambled Control siRNA with the PEQLAB total RNA isolation kit , followed by reverse transcription to cDNA, performed in a thermal cycler (Peqlab) using the high-capacity cDNA reverse transcription kit . The qPCR reaction was set up with the GoTag2+ imaging was performed on a digital wide field microscope, the iMIC  equipped with a 40× objective  and an ultrafast switching monochromator, the Polychrome V (Till Photonics). Illumination of GEM-GeCO1 targeted sensors was performed at 430 nm excitation and emissions were collected with a dichrotome dual emission filter set . pH calibration was performed as described previously  and images were recorded with a charged-coupled device (CCD) camera . Data acquisition and control of the digital fluorescence microscope was performed using the live acquisition software version 2.0.0.12 (Till Photonics).Caeviously  in a Ca21–140-mRuby, CVsue-mRuby or ROMO1-mRuby were loaded with 0.5 μM MitoTrackerTM Green  in storage buffer for 40 min, once washed and imaged in 2CaNa with a super-resolution CFI SR Apochromat TIRF 100x-oil (NA 1.49) objective mounted on a Nikon-Structured Illumination Microscopy (N-SIM®) System with standard wide field and SIM filter sets and equipped with two Andor iXon3® EMCCD camera mounted to a Two Camera Imaging Adapter . Co-localization of MTG fluorescence intensities with MICU11–140-mRuby, CVsue-mRuby and ROMO1-mRuby in the CM and IBM were measured via line scan analysis within a defined rectangle area. Normalized sum of intensities profiles derived from green (MTG) and red (mRuby) fluorescence were calculated to define the full width at half maximum (FWHM) as a measurement for the label latitude. MTG-staining was used as an internal reference for the IMM to determine the distance variation of sub-mitochondrial targeted mRuby at basis of the FWHM.Structured Illumination Microscopy experiments were performed as described previously . In briepost hoc test. The acquired data of Ca2+ measurements were analyzed by the GraphPad Prism software version 5.01 . Data are presented as mean ± standard error of mean (SEM) of independent experiments (n) throughout the whole manuscript. For comparisons between two groups, two-tailed Student t-test was used for evaluation of statistical significance and a P value between 0.01 and 0.05 (p Student’s t-test) was considered significant and indicated with “∗”, P between 0.001 and 0.01 as very significant with “∗∗,” and P < 0.001 as highly significant with “∗∗∗.” For comparisons across multiple groups, one-way ANOVA with Bartlett’s test for equal variances and Bonferroni’s Multiple Comparison test were used for evaluating statistical significance expressed as described above.Statistical analysis for SIM experiments were performed using one way ANOVA and Bonferroni’s 1–140-mRuby were subsequently loaded with the IMM specific dye MitoTrackerTM Green (MTG). Super-resolution structure illumination microscopy (SIM) revealed that MICU11–140-mRuby was exclusively directed to the inner boundary membrane (IBM)  is a specific IMS protein via its N-terminal targeting sequence . Accordine (IBM) . For tarne (IBM) . Moreovene (IBM)  to strucne (IBM) . SIM dathickness . Using tartments .2+ biosensors including the ratiometric GEMGeCO1 (2+ levels (3-mediated Ca2+ mobilization by histamine (100 μM) was more than 50% higher by using the IMS specific MICU11–140-GEMGeGO1 in comparison to both CL targeted probes compartments. First, we tested the suitability of the biosensors for simultaneous measurements by co-expressing MICU11–140-GEMGeCO1 with MICU11–140-CARGeCO1 and alternately recorded the respective emission. Since both biosensors exhibit a similar kD for Ca2+, i.e., 340 nM for GEMGeCO1  or the mitochondrial matrix-targeted (mt-CARGeCO1) red Ca2+ biosensor and imaged the responses upon histamine stimulation and store-operated Ca2+ entry within individual cells. Targeting of the various sensors is illustrated in 2+ measurements simultaneously with cytosolic Ca2+ signals (CARGeCO1) are presented in 2+ with the newly targeted Ca2+ sensors for the IMS, and for both CL probes were shown in 2+ signaling of the very sub-compartment of the mitochondria. Hence, co-imaging of IMS with cytosolic Ca2+ in a fast temporal sampling mode revealed almost simultaneous occurring Ca2+ increase  sub-mitochondrial Ca2+ measurements. We aimed to establish such biosensors for sub-mitochondrial Ca2+ measurements in order to allow the examination of spatial, sub-organellar Ca2+ kinetics that will foster our attempts in investigating mitochondrial Ca2+ homeostasis. Besides the identification of suitable signal sequences, targeting was assessed using super-resolution structural-illumination microscopy (SIM), and a comparative characterization of the very Ca2+ sensitivity was performed. Hence, our work also provides initial (simultaneous) measurements where we applied the new sub-mitochondrial Ca2+ sensors for analyses of the Ca2+ kinetics upon given intracellular Ca2+ signals and, thus, tested the suitability of these new Ca2+ sensors to reveal distinct sub-mitochondrial Ca2+ signals under various conditions of altered mitochondrial Ca2+ signaling.In this study we identified specific sequences that allow the distinct targeting of genetically encoded Ca2+]IMS  and reactive oxygen species modulator 1 (ROMO1) that has been shown to localize exclusively in the CL  sensors exclusively to the CL.So far, only few attempts to target fluorescent indicators to these very sub-mitochondrial regions were successful. In an early study, the cDNA of glycerolphosphate dehydrogenase (GPD) was fused to HA1-tagged aequorin to study [Ca2+]IMS  or [H+]IS  to get initiated (2+ uptake complex (MCUC) is much lower than that in any other mitochondrial sub-compartment, thus indicated some further processes necessary for MCUC activation. This assumption is further supported by the findings that upon a slow Ca2+ release by SERCA inhibition or when Ca2+ flux through the OMM is reduced by knockdown of VDAC1, no or only very small matrix Ca2+ signals occur despite the Ca2+ signals in the IMS is much less affected. Our findings that the prevention of MCUC by knockdown of MCU and EMRE did not affect the Ca2+ signal in the intermembrane space but considerable increased Ca2+ accumulation in the CL point to an active contribution of the cristae to mitochondrial Ca2+ homeostasis.In course of the functional testing of the newly targeted GEMGeCO1 Caironment . By combironment  with difironment  we estabnitiated . However2+ sensors to sub-compartments of the mitochondria and established simultaneous measurements of two distinct locations. Hence, the newly targeted Ca2+ sensors described herein have been approved to be suitable for analyses of sub-mitochondrial Ca2+ signaling and mitochondrial Ca2+ homeostasis, thus, providing suitable tools to discriminate between malfunction of OMM and/or IMM Ca2+ import e.g., during generation of neurodegenerative disease and will foster our understanding on mitochondrial Ca2+ homeostasis in future work.Altogether, in the present work we have successfully targeted well-known CaThe raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.JR-M, MD, CK, EE, HB, SB, and MW-W performed design and construction of sensors. JR-M, CK, and MW-W performed cloning and delivered plasmid material. MW-W and GZ performed imaging experiments and data analysis. CM-S performed qRT-PCR experiments including analysis. BG performed super-resolution SIM experiments and analysis. MW-W together with RM, BG, and WG supervised the project, performed data interpretation and wrote the manuscript. All authors discussed the results and commented on the manuscript at all stages.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Activity of RyR2 and SERCa2a in myocytes is not only dependent on expression levels and interacting accessory proteins, but on fine-tuned regulation via post-translational modifications. In this paper, we review how aberrant changes in intracellular Ca2+ cycling via these proteins contributes to arrhythmogenesis in the aged heart.Aging of the heart is associated with a blunted response to sympathetic stimulation, reduced contractility, and increased propensity for arrhythmias, with the risk of sudden cardiac death significantly increased in the elderly population. The altered cardiac structural and functional phenotype, as well as age-associated prevalent comorbidities including hypertension and atherosclerosis, predispose the heart to atrial fibrillation, heart failure, and ventricular tachyarrhythmias. At the cellular level, perturbations in mitochondrial function, excitation-contraction coupling, and calcium homeostasis contribute to this electrical and contractile dysfunction. Major determinants of cardiac contractility are the intracellular release of Ca Heart disease remains the leading cause of death in the US , in part2+ homeostasis i) activate contractile machinery, which initiates cardiac systole. Relaxation and diastole ensue when Ca2+ is extruded from the cytosol via the Na+/Ca2+ exchanger  and sequestered into the SR via the sarco/endoplasmic reticulum Ca2+-ATPase . Any perturbations in intracellular Ca2+ handling can, therefore, alter contractility and electrical stability of the heart. It is well established that excitation-contraction coupling becomes dysfunctional with age, which drives increased propensity for cardiac arrhythmias in the elderly population i, known as a Ca2+ spark [2+-induced Ca2+ release (CICR) from the SR across the cardiomyocyte [2+ sparks results in a global increase in [Ca2+]i, or Ca2+ transient, which initiates muscle contraction.Much of the intracellular Cael, RyR2 . Within 2+ spark , activatomyocyte . The sum2+ release events during diastole known as SR Ca2+ leak i must be sufficiently reduced for muscle relaxation. Sequestration of Ca2+ from the cytosol back into the SR occurs via SERCa2a, which is an ATP-dependent process that restores SR Ca2+ content. The affinity of SERCa2a for Ca2+ and, thus, its pumping activity is negatively regulated by association with inhibitory protein phospholamban (PLB). Phosphorylation of PLB by PKA or CaMKII relieves this inhibition, which enhances SERCa2a Ca2+ affinity and stimulates SR Ca2+ uptake  in heart failure also enhances reverse mode activity, which may increase [Ca2+]i and, thus, RyR2 activity. It is well established that aging also prolongs action potential and diastole  and [Na+], advancing age in ventricular myocytes of male rats (27–31 months old) was associated with increased forward NCX1 activity, which contributes to prolongation of action potential duration and arrhythmogenesis  ([Ca2+]m). Both increased and decreased [Ca2+]m are reported as deleterious to cardiac function [2+]m, including harsh procedures to isolate mitochondria, or loading myocytes with membrane-permeable dyes that can impair membrane integrity. The development of mitochondrial-targeted genetic Ca2+ probes and in vivo delivery methods should help resolve ongoing controversies  and, thereby, limit deleterious mitochondrial ROS production in the senescent myocardium.Diminished mitochondrial function and enhanced ROS emission has been attributed to changes in the mitochondrial matrix m, and there is evidence that mitochondrial Ca2+ cycling is altered in cardiac disease [2+]m holds therapeutic potential remains controversial [2+ to cardiac excitation-contraction coupling [2+ accumulation augments RyR2 function and SR Ca2+ leak via increased ROS emission [2+ leak via RyR2 directly modulate [Ca2+]m and mitochondrial function? Using phosphomimetic mutations of Serine-2808 to modulate RyR2 function, Santulli et al. (2015) suggested that [Ca2+]m increases as a result of SR Ca2+ leak results in mitochondrial dysfunction in mice [2+ is likely to be taken up by mitochondria [2+ uniporter (MCU) complex to Ca2+ is not thought to be affected by aging [2+ homeostasis in the senescent heart.An alternative approach to modulating redox balance in aged myocytes is the normalization of mitochondrial Ca disease ,149,150.oversial ,167,168,coupling ,172,173.emission , there a in mice . The topchondria , so how by aging , and as 2+ cycling [2+ spark frequency, CaMKII-mediated RyR2 phosphorylation at Serine-2814, and Ca2+ alternans in a dog model of sudden cardiac death, which reduced ischemically-induced VF [2+ spark activity in left ventricular myocytes [2+ homeostasis remains limited. There is some evidence that exercise training improved Ca2+ cycling and contractility, which is associated with increased expression of SERCA2a in aged rodents (28 months old) [The impact of exercise and physical activity on the progression of cardiac aging is also an active area of research. There is evidence in both healthy animal models and models of other cardiac disease phenotypes that exercise can induce alterations in cardiac Ca cycling ,176,177.duced VF . Long-teths old) , while oths old) ,180. In ths old) ,182. Twoths old) . There iths old) ,185. Howths old) ,187,188.2+ handling proteins and arrhythmogeneses in the aged heart, but this is worthy of future investigation. Calorific restriction and fasting remains the only strategy demonstrated to significantly increase one’s health span and lifespan, both in animal models and humans . The eff