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It is now more clear that organelles with double membranes, such as mitochondria, chloroplasts and nuclei can generate calcium signals on their own (Xiong et al. 2006). Already during the 1980th it was shown that light induces a calcium influx into chloroplasts of both wheat and spinach (Muto et al. 1982, Kreimer et al. 1985). Kreimer et al. (1985) suggested that the influx across the envelope of intact chloroplasts was linked to the photosynthetic electron transport. Later on Shabala and Newman (1999) reported, by use of ion-selective vibrating micro electrodes close to the leaf surface, that light also induced changes in H+, K+, Cl- and Ca2+ concentrations in the mesophyll of beans. These ion fluxes were related to changes in plasma membrane potential and calcium influx was the main depolarizing agent in the electrical response to light. Calcium influx started within 5 s, while net fluxes of H+, K+, Cl- did not begin until after 2 min. The initial alkalinization found in the medium was suggested to depend on CO2 uptake by the photosynthesizing tissue, while the activation of the H+-pump occurred 1.5 to 2 min later. Two different mechanisms for uptake of Ca2+ into chloroplasts were suggested; one by a potential-stimulated uniporter at the inner-envelope membrane and another by a H+/ Ca2+ antiporter in the thylakoids fueled by ATP (Xiong et al. 2006).
Elevated temperatures induce many changes in gene expression leading to thermotolerance and improve the cell survival to high temperature. Results from Gong et al. (1998) suggest that cytosolic Ca2+ also is involved in heat-shock (HS) signal transduction. By use of transgene tobacco where the aequorin protein was targeted both to the cytosol and to chloroplasts, they could show that HS induced a prolonged, transient increase of Ca2+ in the cytosol, but not in the chloroplasts. Inhibitor analyses suggested that Ca2+ was mobilized from both intracellular and extra-cellular sources. There are some reports showing that the chloroplast can control the cytosolic Ca2+-transients involved in stomatal closure (Nomura et al. 2008, Weini et al. 2008).
3.2.3 Calcium signaling and transport in mitochondria
Logan and Knight (2003) described the first successful targeting of aequorin to plant mitochondria. They found that the resting values of free Ca2+concentration differed in the cytosol and in the mitochondria. In the cytosol of Arabidopsis this value is around 100 nM, but in the mitochondria around 200 nM. Treatments of Arabidopsis seedlings floating in water, with cold, osmotic (mannitol), touch, and oxidative (hydrogen peroxide 10 mM) stresses all showed almost the same calcium signature (kinetic pattern) in both cytosol and mitochondria. However, except for hydrogen peroxide addition, the amplitude was much higher in cytosolic, than in mitochondrial calcium increase. Touch stress induced an immediate very high elevation, followed by a return to near-resting concentration within 20 s in the cytosol, but in the mitochondria the return to resting level was much slower. All the other stresses induced more prolonged reactions. The addition of hydrogen peroxide caused almost the same reaction in both compartments and the results, therefore, indicated that mitochondria could be more sensitive to oxidative stress than to the other stresses.
Calcium and palmitic acid (Pal) induced a stable and prolonged partial depolarization of the mitochondrial membrane, pore opening, release of calcium and swelling of mitochondria (Mironova et al. 2007). Addition of inhibitors of Ca2+ uniporters, like ruthenium red and La3+, as well as EGTA, added 10 min after Pal/Ca2+-activated pore opening, prevented the release of Ca2+ and re-polarized the membrane to initial level. The authors also found similar effects in mitochondria accumulating high strontium, Sr2+, concentration in the absence of exogenous Pal, which lead to activation of phopholipase A2 and formation of endogenous fatty acids. They concluded that Ca2+ is taken up into mitochondria by a uniporter and that Ca2+ efflux is mediated by a short-living Pal/Ca2+-activated pore. Under oxidative stress an increase of electron transport in mitochondria triggers H2O2 production, depletion of ATP, opening of the permeability transition pores (PTP) and cell death (Tivari, Belenghi and Levine 2002).
3.2.4 The nucleus can generate and regulate calcium signals on its own
The nucleus can be considered as two compartments: the nucleoplasm and the nuclear envelope, in which calcium is stored. The envelope lumen is connected with the lumen of the endoplasmatic reticulum (Bach, Agell and Carafoli 1992). Probably both the envelope and the nucleoplasm have a calcium buffering capacity (Briere et al. 2006). The envelope has pores that allow molecules up to 40 kDa to penetrate (Brandizzi, Irons and Evans 2004). It is well known that calcium-dependent processes take place in the nuclei (Xiong et al. 2006). For instance, an increase in free calcium level is necessary for activation of nucleur sensing kinases and phosphatases in the nucleus and other steps in the signaling pathways both in animals (Carafoli 2002), as well as in plants (Harper, Breton and Harmon 2004). It has been suggested that the nucleus can be described as "a cell within the cell" (Bkaily 2006, Gomes et al. 2006).
Since 1999 it was possible to detect free calcium concentration changes in the nucleus, [Ca2+]n. Van der Luit (1999) succeeded to fuse aequorin to nucleoplasmin, a nuclear protein. They investigated effects by wind and cold shock on both cytosolic and nuclear changes of calcium in tobacco seedlings, and found different dynamics in [Ca2+]n and [Ca2+]cyt. A simultaneous rapid increase of both [Ca2+]cyt and [Ca2+]n, were obtained, but the increase in [Ca2+]n, was delayed with respect to the cytosolic changes.Wind and cold also induced the expression of a calmodulin gene (NgCAM-1) and a comparison between calcium dynamics with gene expression indicated that wind-induced expression depends on nuclear calcium signaling, while cold shock-induced expression is mediated by cytosolic calcium elevation.
When tobacco seedlings were subjected to decreased osmolarity of the culture medium, an increase of [Ca2+]cyt was followed by an increase of [Ca2+]n but the Ca2+]n remained for much longer time than the [Ca2+]cyt (Pauly et al. 2001). On the other hand, increase of the osmolarity of the culture medium elicited a smaller change in [Ca2+]cyt, but did not modify the biphasic shape of the cytosolic response, and did not affect the [Ca2+]n.
Bimodal long-lasting changes in [Ca2+]cyt were also found when proteinaceous elicitors were used (Lecourieux et al. 2005). Cryptogein, a polypeptidic elicitor induced a transient peak in [Ca2+]cyt after 5 min and then a a sustained increase for at least 2h. On the other hand, a weak increase in [Ca2+]n was followed by a substantial increase for 1 h, but with maximum at 23 min and, thus, peaked much later than the [Ca2+]cyt. It was suggested that a 1-s delay between the cytosolic and nuclear responses is enough to exclude the possibility of just a diffusion of Ca2+ from the cytosol to the nucleus of the cell (Meyer, Allbritton and Oancea 1995). Xiong et al. (2008) showed that sphingolipid metabolites selectively elicited increase in nuclear calcium concentrations in a dose-dependent manner both in cell suspensions and in isolated nuclei of tobacco BY-2 cells. Thus, the nuclear calcium changes were independent of the cytosolic compartment.
In pollen tubes calcium gradients in the tip were oscillating in the cytosol during normal polarized growth, but no measurable changes in [Ca2+]n were found (Watashiki, Trewavas and Parton 2004). Thus, the available results so far demonstrate that different calcium signals proceed in different cell compartment and that the [Ca2+]n is not directly linked by diffusion to a change in [Ca2+]cyt (Mazars et al. 2009).
Proteins that are important for the amplification or diminishing of calcium signals are found to shuttle to and from the nucleus. Rodriguez-Concepcion et al. (1999) showed that calmodulin (CaM) 53 could be localized reversible to the nucleus to the plasma membranes. When localized to the plasma membrane it was isoprenylated at the C-terminus domain, but inhibition of isoprenoid biosynthesis caused an accumulation of CaM to the nucleus. They also showed that CaM was associated with the plasma membrane after light exposure and was transferred to the nucleus in darkness. It has been suggested that also other calcium-binding or CAM-binding proteins, as well as calcium-dependent enzymes and different transcription factors are present in the nucleus and regulate the nucleus activity (Mazars et al. 2009). Calcium signals may also be generated via G-coupled receptors localized to the nucleus without involvement by the plasma membrane. It is suggested that the nucleus can produce inositol 1,4,5 triphosphate (IP3) and IP3 receptor-mediated Ca2+ release (Gomes et al. 2006). Nuclear activities are probably regulated by a cross-talk between ROS and calcium (Mazars et al. 2010).
4. Calcium signaling under anoxia
Higher plants are strict aerobic organisms, which directly depend on molecular oxygen for their respiration and other metabolic processes. Nevertheless, very often they suffer from oxygen shortage in different agricultural, horticultural and industrial areas. Availability of oxygen has a strong influence on distribution of plant species in ecosystems and severe economical impact. Yield loss due to deficiency of oxygen could reach up to 50% (Dennis et al. 2000). The lack of oxygen usually results from excess of water, ice crust and is due to soil compaction. Waterlogging of rhizosphere and partial flooding of aboveground parts of plants lead to gradual hypoxia (deficiency of oxygen) and complete submergence brings about anoxia (total absence of this gas).
4.1 Anoxic injury in plants
The lack of molecular oxygen leads to inhibition of aerobic respiration, which in turn ultimitly causes energy starvation. After the switch from aerobic condition, ATP level in the cell exhausts within 1-2 min (Drew 1997). To provide ATP for energizing cellular metabolism glycolysis is passed into fermentation by activation. Overconsumption of respirable sugars by these processes aggravates energy starvation, particularly in hypoxia-susceptible species (Vartapetian and Jackson 1997) . On the other hand, stimulation of lactic fermentation and lack of ATP to energize transport ATPases account for cytosol acidification (Drew 1997). Alcoholic fermentation generates a toxic intermediate (acetic aldehyde) and end-products (ethanol), that together with acidosis self-poison plant under oxygen deficiency (Vartapetian and Jackson 1997, Gibbs and Greenway 2003, Bailey-Serres and Voesenek 2008). In natural inhabitates flooding leads to low soil-redox potential and production of reduced substances including Mn2+, Fe2+, H2S, NO2- and intermediates of carbon metabolism such as methan, ethan, ethylene, acethylene, acetic and butyric acid ( Drew 1997 and references wherein). Moreover, reestablishment of normoxic conditions triggers oxidation of these substances and synthesis of reactive oxygen species (ROS) resulting in post-anoxic injury.
4.2 Oxygen deficiency tolerance mechanisms in plants
Capacity to survive oxygen deprivation depends on a number of developmental, morphological and metabolic adaptations in plants. Imposition of hypoxia accelerates growth of shoot axial organs, stimulates formation of adventitious roots and aerenchyma particularly in tolerant plant species. As a result, the shoot actively transports oxygen to a flooded root (for review see Crawford and Braendle 1996, Drew 1997, Vartapetian and Jackson 1997, Kende, Van Der Knaap and Cho 1998, Sauter 2000, Gibbs and Greenway 2003, Bailey-Serres and Voesenek 2008). Simultaneous shifts occur in the metabolism, which are particularly severe under strict oxygen lack. Metabolic adaptations include mainly avoidance of energy starvation, prevention of toxicity of anaerobic intermediate and end products, and post-anoxic injury, disposal of cytosol acidification and use of alternative electron acceptors (like nitrate, nitrite, unsaturated lipids, etc.) (Crawford and Braendle 1996, Drew 1997, Vartapetian and Jackson 1997, Gibbs and Greenway 2003, Greenway and Gibbs 2003, Bailey-Serres and Voesenek 2008). Hypoxia-tolerant plants are notable for maintenance of their cell ultra structure (Vartapetian and Jackson 1997), membrane stability (Crawford and Braendle 1996) and synthesis of anaerobic stress proteins (Vartapetian and Jackson 1997; Greenway and Gibbs 2003). Most of the anaerobic stress proteins belong to enzymes of the glycolytic or fermentative pathways, carbohydrate mobilisation and nitrogen metabolism ( Vartapetian and Jackson 1997, Greenway and Gibbs 2003). All together these metabolic adaptations allow tolerant plants to generate sufficient amount of energy, maintain mineral uptake and even to grow in total absence of oxygen.
4.3 Perception of anoxic stress in plants
The mechanism(s) of oxygen sensing by plant cell and sensor itself are still to be elucidated. Oxygen depletion could be detected directly by binding molecular oxygen (direct sensing) or recognized by altered cellular metabolism (indirect sensing) ( Bailey-Serres and Chang 2005, Licausi and Perata 2009) . Only prokaryotes have direct oxygen sensors, including haem-containing protein kinases, Fe/S cluster- and SH-containing transcription factors, which induce anaerobic gene expression and are involved in aerotaxis ( Green et al. 2009). None of the eukaryotes is reported to possess direct mechanism of oxygen sensing. In fungi and animals anaerobic genes are regulated at transcriptional level. Hap-transcription factors are involved in oxygen sensing in yeast and hypoxia-inducible factor (HIF) is widespread throughout animal kingdom ( Bailey-Serres and Chang 2005, Bailey-Serres and Voesenek 2008, Licausi and Perata 2009) . Activity of these factors is regulated by the oxygen level, production of ROS and redox state of the cell. Intracellular pH, ATP and sugar level are good candidates for indirect sensing signal too.
4.4 Cytosolic calcium signaling in plants under anoxia
The main effect of anoxia sensing in eukaryotic cells is an increase in [Ca2+]cyt. There are two major hypotheses explaining this reaction in animal physiology ( Lahiri et al. 2006, Fâ€žhling 2008) (Fig 1) . The "mitochondrial" hypothesis states that the mitochondrial electron-transport chain is retarded by depletion of oxygen, produces ROS that leads to a Ca2+ release into the cytosol from the mitochondria and other intracellular compartments. The "plasma membrane" or "NAD(P)H-oxidase" hypothesis concerns involvement of ROS generated by NAD(P)H-oxidase. This leads to suppression of outward plasmalemma K+-channels, a depolarization of the membrane and a Ca2+ influx from extracellular Ca2+ stores.
Subbaiah, Bush and Sachs (1994a) first reported elevation of [Ca2+]cyt in maize suspension-cultured cells, which was completely reversible after a few seconds of reoxygenation. Treatment with Ca2+ -channel blockers prevents anoxic induction of ADH1 and SuS1 genes in plant cell and post-anoxic seedling survival (Subbaiah, Zhang and Sachs 1994b ; Subbaiah 2009). In maize cells the elevation of [Ca2+]cyt within the first minutes of anoxia derives from efflux of Ca2+ from intracellular stores, as it is significantly inhibited by ruthenium red (RR, inhibitor of intracellular membrane Ca2+/H+-antiporter) , but not affected by EGTA (Ca2+ chelator) and various inhibitors of plasma membrane Ca2+ permeability ( Subbaiah, Bush and Sachs 1994a ) . Moreover, subsequent research showed that elevation of Ca2+ in the cytosol of maize cell originates from mitochondria (Subbaiah, Bush and Sachs 1998) and could be induced by production of ROS by the mitochondrial electron transport chain under lack of oxygen according to "mitochondrial" hypothesis ( Rhoads et al. 2006 ).
On the other hand, a transient spike and prolonged elevation of [Ca2+]cyt in transgenic apo-aequorin-expressing Arabidopsis seedling upon imposition of anoxia depends on external as well as internal stores, since the downstream anaerobic gene expression is partially inhibited by EGTA, Gd3+, La3+ (non-specific inhibitors of plasma membrane Ca2+ channels) and RR (Sedbrook et al. 1996) . This supports both the hypotheses. Our recent results (Yemelyanov et al. prepared for publication) showed ultimate importance of both external and internal Ca2+ stores for anoxic signaling in rice, whereas the hypoxia-intolerant wheat does not require external sources for that purpose.Â
Ca2+ was shown to be important for anoxic induction of ADH in Arabidopsis and rice seedlings (Dolferus et al. 1997, Tsuji et al. 2000). Aurisano, Bertani and Reggiani (1995, 1996) demonstrated involvement of Ca2+cyt, plasma membrane Ca2+-channels, CDPKs, CaM and G-proteins in anoxic signal transduction leading to accumulation of GABA and other amino acids in rice.
Influx of Ca2+ via plasma membrane channales in Arabidopsis and rice is closely related with production of ROS on the cell surface. Blokhina, Chirkova and Fagerstedt (2001) revealed production of ROS in the apoplast of different monocot species under anoxia. In Arabidopsis seedlings induction of ADH gene depends on H2O2. Moreover, treatment of seedlings with diphenyleneiodonium, an inhibitor of ROS production by NAD(P)H-oxidase, blocked hypoxia-induced ADH activation (Baxter-Burrell et al. 2002). This links ROS production by NAD(P)H-oxidase, ADH induction and Ca2+ signaling.
Scheme of Ca2+ signaling in plants under anoxia is shown on the Fig. 1. In maize and wheat plants accumulation of [Ca2+]cyt occurs mainly via "mitochondrial" pathway, whereas in Arabidopsis and rice it passes through both, "mitochondrial" and "plasma membrane" pathways together.
5. Cold temperature stress signaling
Many plant species have the possibility to enhance freeze tolerance after exposure to non-freezing temperatures. Cold acclimation is associated with alterations in plasma membrane lipid composition, increase in proline and sugar contents, synthesis of new polypeptides and changes in the mRNA populations (Steponkus and Lynch 1989, Guy 1990, Lin et al. 1990, Monroy, Sarhan and Dhindsa 1993, Uemura, Joseph and Steponkus 1995).
5.1 Cold-induced changes in plasma membrane lipid composition
The plasma membrane lipid composition is changed under cold stress in order to stabilize the membranes against freeze injury. Acclimation results in more unsaturated fatty acids, which cause a drop in the transition temperature. In Arabidopsis maximal freeze tolerance was induced after one week at 2Â°C (Uemura, Joseph and Steponkus 1995). During that time the proportions of phospholipids in the plasma membranes increased, while cerebrosides and free sterols decreased. Moreover, the di-unsaturated species of phophatidylcholine and phosphatidylethanolamine increased. Some proteins, like dehydrins and lipocain may help the plant to prevent damage to the plasma membrane during freezing (Uemura et al. 2006).
5.2 Calcium is involved in the acclimation to cold temperature
Calcium has a role in cold-induced changes in protein phosphorylation, gene expression and development of freeze tolerance (Monroy, Sarhan and Dhindsa 1993, Knight, Trewavas and Knight 1996, Thtiharju et al. 1997, Viswanathan, Zhu and Zhu 2006). In the freeze-tolerant alpine plant Chorispora bungeana, chilling induction at 0øC increased the calcium contents (Fu et al. 2006). The Ca2+ levels were different in various tissue and organs. Knight, Trewavas and Knight (1996) reported that inhibition of calcium influx caused a partial inhibition of cold-dependent kin1 expression in Arabidopsis. It was shown that inhibitors of calcium channels, calmodulin action or protein kinases, also inhibited development of freezing tolerance. In alfalfa plants calcium elevation caused by low temperature induced expression of two cas (cold acclimation-specific) genes cas15 and cas18 (Monroy and Dhindsa 1995). In Arabidopsis cold stress induced the kin1 and kin2 genes but the kin2 mRNA accumulated to a higher level than the kin1 mRNA under acclimation (Kurkela and Franck 1990). Many cold-regulated genes have in their promoters one or several copies of the DRE/CRT cis-element (DEHYDRATION RESPONSIVE ELEMENT/C repeat). Other transcription factors bind to this element and activate transcription of downstream genes (Zhu 2001). The CBF/DREB1 genes are also induced directly by a low temperature and the induction precedes that of genes containing the DRE/CRT cis-element. Thus, a network of multiple signaling pathways are involved in cold stress response in plants, and some of them are also induced by salt and drought stresses in a complicated way. It was proposed by Zhu (2001) that low temperature specifically induces the transcription of the CBF/DREB1-based cascade. Calcium is required for full expression of the CRT/DRE controlled COR6 and KIN1 genes of Arabidopsis (Monroy and Dhindsa 1995, Knight, Trewavas and Knight 1996). In the regulation of of cold-responsive genes and freezing tolerance also a CBF-independent pathway exists (Viswanathan, Zhu and Zhu 2006). The transcription factors HOS9 (a homeodomain type) and HOS10 (a myeloblastosis type) play an important role in this pathway.
Calcium is important not only for gene expression during cold acclimation, but also has an effect on resealing the membranes after cold stress (Yamasaki et al. 2008). In experiments with Arabidopsis protoplasts extra-cellular calcium increased tolerance to electroporation, which punctures the membrane. An antibody against a homolog of synaptotagmin, SYT1, inhibited the calcium-dependent freezing-tolerance in Arabidopsis leaf protoplasts. This substance is known to be a calcium sensor that causes exocytosis. This inhibition indicates that the puncture allowing the antibody to enter into the cytoplasm occurs during freeze/thawing. The authors suggested that the calcium-dependent freezing tolerance results from membrane resealing and that SYT1 is involved in this mechanism.