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In transgene Arabidopsis, in which aequorin was targeted to different types of root cells, as well as to the whole plant, cold-water addition caused a transient rise in [Ca2+cyt] (Kiegle et al. 2000). The maximum peak occurred at approx. 15-20 s in all cell types. However, the increase in [Ca2+cyt] was lower in the root cells, compared with shoot cells. The largest elevation was obtained in the deepest cell type, the pericycle, and the lowest peak level in the elongation zone. Therefore, it was concluded that the size of the perceived agonist does not decide the magnitude of the calcium response. Instead, there is a cell-specific response.
Calcium dynamics under chilling stress were also investigated in single mesophyll protoplasts from tomato plants loaded with the calcium specific dye Fura2, AM (Sebastiani, Lindberg and Vitagliano 1999). The protoplasts were subjected to chilling stress (10-15°C) in a temperature-controlled perfusion chamber. The results showed that different kinetics in [Ca2+cyt] occurred depending on different resting-calcium levels. In 84 % of the investigated protoplasts there was an increase in [Ca2+cyt]. In 21% of the reacting protoplasts, the maximum [Ca2+cyt] was obtained after 10-20 s, thus corroborating results from root cells of Arabidopsis (Kiegle et al. 2000); in 11 % of the protoplasts both the increase and decrease in [Ca2+cyt] were slower; and in 32 % a constant increase of [Ca2+cyt] was obtained 1 min after start of temperature decrease. When the resting calcium concentration was higher than normally, the increase in [Ca2+cyt] was constant. In these experiments the cooling rate was constant because a perfusion system was used. Therefore, it is likely that different cells have different competence and ability to maintain calcium homeostasis. A sustained high [Ca2+cyt] is implicated in apoptosis, and in hypersensitive responses to pathogens (Levine et al. 1996).
When a cold solution (5øC) was added to mesophyll protoplasts of wheat leaves, much larger changes in [Ca2+cyt] were obtained than in chilling sensitive tomato protoplasts, in which a perfusion system was used (Lindberg, Sebastini and Vitagliano 1999, Fig 2). A second addition of cold solution to the same wheat protoplast induced different Ca2+cyt kinetics. The first addition of cold solution gave a transient prolonged increase of [Ca2+cyt], which was much smaller than that induced by a second addition (Figure 2A,B). The second addition gave a peak response and, thereafter, a prolonged calcium increase (Fig 1B). These results cannot be compared with results shown by Knight, Trewavas and Knight (1996), since these authors report a mean of 3 or more cold additions at time zero. When the wheat protoplasts were treated with erythrosine B, an inhibitor of Ca2+ATPase in the ER and plasma membrane, the calcium transient was prolonged and the magnitude was much higher than without the inhibitor (Fig 2C). Therefore, it is likely that the Ca2+ATPase is involved in the signaling by pumping Ca2+ out from the cytosol.
It has been proposed that the [Ca2+cyt] signatures are modified by previous experience, which means that the plant has a calcium "memory" (Knight, Trewavas and Knight 1996). The magnitude of [Ca2+cyt] elevation elicited by wind becomes progressively smaller upon repeated stimulation and for some stimulus several hours are needed for a second reaction to take place (Tuteja and Mahajan 2007). The calcium signature can also be modified during a second exposure to a stress as shown in Figure 1. Moreover, the magnitude of [Ca2+cyt] increase can be changed by prior in vivo exposure to a contrasting stress. These observations imply a cross talk between the signaling cascades.
Cold-shock responses were compared in chilling-sensitive tobacco and chilling-tolerant Arabidopsis (Knight, Trewavas and Knight 1996). In both cases an immediate rise in [Ca2+cyt] was obtained. In Arabidopsis both EGTA and lanthanum caused a partial inhibition of [Ca2+cyt] and of cold-dependent kin1 gene expression. To investigate if the vacuole was involved in the calcium signaling, aequorin was targeted to the cytosolic face of the tonoplast. The results showed that a higher peak of calcium elevation was obtained in the cytosol than in the tonoplast micro-domain. In addition, the elevation in the microdomain, [Ca2+mic], was maintained for a longer time than the [Ca2+cyt]. Pretreatment with neomycin or lithium, which interferes with phophoinositide cycling, diminished the calcium reactions, showing that some efflux of calcium occurred from the vacuoles. The magnitude of the cold-shock induced [Ca2+cyt] response was similar in Arabidopsis and tobacco, but was more prolonged in tobacco. Upon repetitive addition of cold solution, the response after 3 and 10 min was weaker in both species. However, tobacco was able to recover its ability to respond fully to cold shock 30 min after the initial shock, whereas Arabidopsis was not. Another difference between the species was that they responded in different way to cold shock after acclimation. Only in chilling-tolerant Arabidopsis cold acclimation altered the signature of [Ca2+]cyt, so that cold shock caused a reduced peak but a prolonged profile. The different mechanisms in stress response could depend on the different sensitivity of Arabidopsis and tobacco to chilling stress. Cold shocks of different amplitude applied to protoplasts of freeze-sensitive olive tree caused transient increases in [Ca2+cyt] which differed in non-acclimated and acclimated protoplasts (D'Angeli, Malho and Altamura 2003). Upon repeated cold-shock treatments the transient increases in [Ca2+]cyt were only reduced when using non-severe rate changes in temperature. In the acclimated protoplasts the [Ca2+]cyt elevations were further reduced.
Temperature sensing in Arabidopsis depends both on the cooling rate (Plieth et al. 1999) and the final temperature to which cooling occurs (Knight 2002). Plieth (1999) presented a mathematical model of how cold is sensed by a plant based on a passive influx across the plasma membrane and an active efflux by a pump. A single peak in [Ca2+cyt] is obtained with a cold shock, that is, at a very fast temperature decrease, but at low cooling rate, the response lacks the initial peak more or less. The pump slows down at low temperature leading to a second slow phase of the increase in [Ca2+cyt]. A biphasic response to a single cooling step is thus obtained when the sensitizing action by the temperature change on the pumps and channels are of equal magnitude (Plieth 1999).
By applying patch clamp technique to Arabidopsis mesophyll cells, Carpaneto et al. (2007) showed that cold induced a rapid increase in [Ca2+cyt], and that the influx of calcium could occur through non-selective cation channels.
5.4 Cold shock induces changes in the membrane potential
A drop in temperature could also change the trans membrane potential. Krol, Dziubinska and Trebacz (2004) showed that the obtained transient depolarization of membrane potential in mesophyll cells of Arabidopsis, Helianthus and Vicea, induced by temperature decrease, depended on calcium influx from both apoplast and internal stores. It was verified later that the cold-induced depolarizations depended on calcium influx (Carpaneto et al. 2007). Verapamil, a calcium channel blocker, caused significant suppression of the cold-induced potential changes. Since the presence of calmodulin antagonists prolonged the repolarization, this could be attributed to activation of CaM-dependent Ca2+-ATPases (Krol, Dziubinska and Trebacz (2004). It was reported that this enzyme is involved in low temperature response in rice (Martin and Busconi 2001). A suggested model for cold signaling in plants is shown in Fig. 3.
6. Salinity; Sodium and osmotic stress signaling
Soil salinity is a major environmental hazard worldwide as more than 40% of the earth is arid or semi-arid and most of the planet's water is saline. Currently more than 6% of the world's land, which exceeds 20% if only the irrigated area is considered, is affected by varying degrees of soil salinity (Flowers and Yeo 1995, Munns 2002, Kader and Lindberg 2008). However, nearly 50 percent of the irrigated land is in the arid and semi-arid regions of the world, and is facing most serious salinity problems.
6.1 Salinity stress has an impact on agricultural productivity
Salinity stress adversely affects agricultural productivity by decreasing the crop yield in many ways. Furthermore, the saline area increases day by day due to sea-level rise and, thus, will have profound harmful effects on agricultural productivity in many countries of the world. Long time ago Buringh (1979) estimated that at least ten hectares of arable land are lost from the agricultural production in every minute, of which three hectares are lost due to soil salinization. In 2002, FAO reported that about 20-30 million hectares of irrigated land were seriously damaged by the build-up of salts and 0.25-0.50 million hectares were to be lost from production every year as a result of salt build-up (Martinez-Beltran and Manzur 2005). The disastrous consequence of this increasing salinity stress together with the growing world population is certainly threatening the future stable global food availability. Agricultural productivity in the future will mostly depend on our ability to identify or develop salt-tolerant crop plants and to grow them in rapidly increasing salt-affected lands.
6.2 Salinity stress injury in plants
The NaCl-dominated salinity in nature imposes two primary harmful effects on plants: one is osmotic stress and the other one is ionic toxicity. Due to the presence of high salt, salinity stress increases the osmotic pressure in the soil solution over the osmotic pressure in plant cells. As a result, plant loses its ability for uptake of water and minerals, especially the uptake of K+ and Ca2+(Glenn, Brown and Kahn 1997, Munns, James and Lauchli 2006). Plant growth inhibition by high amounts of Na+ and Cl- is one of the main deleterious effects of salinity stress. When present in excess amount, Na+ and Cl- ions can enter into the plant cells and exert toxic effects on cell membranes, and on metabolic activities in the cytosolic part of the cell (Greenway and Munns 1980, Hasegawa et al. 2000, Zhu 2001). The resultant effect of osmotic stress and ionic toxicity may lead to secondary effects in plants such as decreased cell expansion, production of assimilate and membrane functions, decreased cytosolic metabolism and raised production of reactive oxygen intermediates (ROSs).
6.3 Salinity stress tolerance mechanisms in plants
As palaeontological and molecular evidence suggest, the embryophytes (terrestrial plants that are not algae) were evolved from the Streptophyta some 500 million years ago (Raven and Edwards 2001). The evolution of salinity tolerance mechanisms in halophytic plants has recently been reviewed by Flowers, Galal and Bromham (2010a). Though physiological foundations of salinity tolerance are present in all plants, plant species show a very wide range of adaptability to salinity stress. For example, glycophytes like chickpea is very sensitive to salinity stress, and suffer toxicity at just 25 mM NaCl (Flowers et al. 2010b), whereas halophytes can tolerate salinity concentration as high as 1000 mM (Khan, Ungar and Showalter 2005). The recent advancement in molecular biology research is uncovering the mechanisms of salinity stress tolerance including the key genes involved in the molecular networks and the signaling cascade that mediates plant responses to salinity stress. Since salinity stress elicits two different adverse effects like osmotic stress and ionic toxicity in plants, plants need different tolerance mechanisms to be adopted under this stress. To deal with the ionic toxicity under NaCl-dominated salinity stress the key mechanisms for tolerance are a diminished toxic ion uptake into the cytosol, ability to limit the entry of these toxic ions into the transpiration stream, the ability to regulate transpiration in the presence of these toxic ions and compartmentalization of Na+ and Cl- ions in to the apoplast, or into the vacuole (Blumwald 2000, Tester and Davenport 2003, Kader and Lindberg 2005, Kader and Lindberg 2008, Munns and Tester, 2008, Flowers and Colmer 2008, Flowers, Galal and Bromham 2010a). Compartmentalization of toxic Na+ into the vacuole is advantageous, since it is no more toxic for the cell, and also a benefit both for growth and for adjustment of the osmotic potential (Flowers and Lauchli 1983, Jhu 2003, Subbarao et al. 2003, Rodr¡guez-Navarro and Rubio 2006 ). Jou et al. (2006) showed that excess Na+ also can be compartmentalized in ER and Golgi bodies.
An important tolerance mechanism is also a plant's capability to increase the uptake of potassium, K+, and to decrease the uptake of Na+ into the cytosol under high sodium concentration. Expression analyses of transporter genes for K+ and Na+ transporters OsHKT1 and OsHKT2 showed that they were differently expressed in tolerant and sensitive rice cultivars (Kader et al. 2006).
To obtain osmotic homeostasis the synthesis of compitable organic solutes, like glycine betaine, mannitol, pinitol, proline, sorbitol, sucrose and trehalose in the cytosol is of importance (Bohnert and Jensen 1996, Chen and Murata 2002, Zhu 2002, Zhang, Creelman and Zhu 2004, Chinnusamy, Jagendorf and Zhu 2005, Taiz and Zeiger 2006, Liang et al. 2009).
6.4 Perception of salinity stress in plants
Cellular perception of salinity stress by plants is prerequisite to start the activation of the whole cell-signaling cascade. This begins with an elevation of [Ca2+]cyt and ends with different tolerance mechanisms activated in the plant. Like other stresses, salinity stress (both osmotic stress and ionic toxicity) is perceived in plants at the cell membrane, either extra-cellularly or intra-cellularly by a protein spanning the plasma membrane and/or an enzyme within the cytosol (Zhu 2003). Under salinity stress, a low K+ level in the cytosol may also lead to cytosolic calcium signals (Luan, Lan and Lee 2009). For sensing the osmotic component of salinity stress,several sensors probably are involved (Urao et al. 1999, Reiser, Raitt and Saito 2003, Tamura et al. 2003, Boudsocq and Lauriere 2005, Tran et al. 2007, Wohlbach, Quirino and Sussmand 2008). A substantial progress in understanding the signal transduction under Na+ toxicity was made by identification of the Salt Overly Sensitive (SOS) pathway in Arabidopsis (Zhu, 2002). In a recent review Luan, Lan and Lee (2009) suggested that CBL (calcineurin B-like proteins) -CIPK (CBL-interacting protein kinase) pathways regulate Na+ transport in plants and thus confer salinity tolerance. The CBLs also appear to be an important group for conferring salinity tolerance through enhanced K+ uptake under salinity stress (Luan, Lan and Lee 2009). As shown in Fig. 4, the salinity tolerance mechanisms in plants entails SOS3, a Ca2+ sensor in the cytosol, that reads the changes in [Ca2+]cyt under salinity stress and specifically binds Ca2+. Then this protein interacts with a SOS2 protein kinase. Thereafter, the SOS3-SOS2 complex in turn activates the plasma membrane Na+/H+ antiporter, the SOS1 protein, and re-establish the Na+ homeostasis of the cells. An elevation of [Ca2+]cyt is also detected by CBL10, which in interaction with SOS2 might trigger tonoplast Na+/H+ antiporter to transport Na+ from cytosol to vacuole. Furthermore, the increase in [Ca2+]cyt can also be perceived by CBL1 and CBL9, which then bind to CIPK23 and interact with the C-terminus of AKT1. In this way the AKT1 channel is activated causing an increased K+ uptake into the cell, which also confers salinity tolerance.
However, it is still necessary to clarify how Na+ toxicity is sensed by the plant cell. It was suggested that the SOS1 protein, with its long C-terminal tail in the cytosol, might sense NA+ (Zhu, 2003, Zhang, Creelman and Zhu 2004, Shabala et al., 2005 ). Kader et al. (2007) showed that for Na+ sensing in rice protoplasts, Na+ first must enter in to the cytosol. The results corroborate the earlier suggestion that the cytosolic tail of the SOS1 protein might sense Na+. On the other hand, in the halophytic plant quince, Na+ entry in to the cell was not necessary for the shift in cytosolic Ca2+ (D'Onofrio and Lindberg, 2009). Therefore, the question still remains to be answered concerning the sensors for Na+ toxicity in plants, and if they differ in different species, and/or in salinity-sensitive and salinity tolerant cultivars.
6.5 Cytosolic calcium signaling in plants under salinity stress
It is evidently clear that the salinity-stress perception, irrespective of how the stress is perceived, triggers an intracellular signaling cascade starting with the elevation of secondary messenger molecules like calcium in the plant cytosol [Ca2+]cyt. Many studies were done to measure the [Ca2+]cyt changes in cells, organs or intact plants under salinity stress by use of different techniques. Fluorescence microscopy measurements were performed in root hairs ( Halperin, Gilroy and Lynch 2003) and in individual mesophyll protoplasts (Kader et al. 2007, D'Onofrio and Lindberg 2009) Measurements in intact whole plants were made by aequorin luminescence detection (Knight, Trewavas and Knight 1997, Gao et al. 2004, Henriksson and Henriksson 2005, Tracy et al. 2008). These studies suggest that the "signature" of [Ca2+]cyt change, e.g. the amplitude, duration and frequency, is very important for transferring of specific downstream reactions leading to stress tolerance.
6.6 The signature of cytosolic calcium [Ca2+]cyt differs
The change in [Ca2+]cyt varies with species, cell type or tissue type and also with different techniques used (Cramer and Jones 1996, Kiegle et al. 2000, Kader et al. 2007, Tracy et al. 2008, D'Onofrio and Lindberg, 2009). The change in [Ca2+]cyt activates different downstream reactions, such as up-regulation or down-regulation of different genes. The reactions also may change with the particular stress (Kiegle et al. 2000), the stress development rate (Plieth et al. 1999), Tracy et al. 2008, D'Onofrio and Lindberg 2009), and pre-exposure to the stress (Knight, Trewavas and Knight 1997) and the tissue type (Kiegle et al. 2000), Tracy et al. 2008). Upon application of 100 mM NaCl to root cells of Arabidopsis (Cramer and Jones 1996, Halperin, Gilroy and Lynch 2003), or to corn root protoplast ( Lynch and Lauchli 1988), a decrease in [Ca2+]cyt was obtained. On the other hand, most studies show an increase in [Ca2+]cyt upon salinity stress (Bittisnich, Robinson and Whitecross 1989, Lynch, Polito and Lâ€žuchli 1989, Knight, Trewavas and Knight 1997, Halfter, Ishitani and Zhu 2000, Kiegle et al. 2000, Knight 2000, Zhu 2001, Gao et al. 2004, Henrikson and Henrikson 2005, Kader et al. 2007, D'Onofrio and Lindberg, 2009
6.7 Plant roots and shoots signal differentially under sodium toxicity and osmotic stress.
Contrasting results are reported whether osmotic stress increases or decreases [Ca2+]cyt (Cramer and Jones 1996; Knight, Trewavas and Knight 1997, Kiegle et al. 2000, Kader et al. 2007). Osmotic and ionic stresses induced different shifts in [Ca2+]cyt in Arabidopsis root cells and the heterogenous [Ca2+]cyt changes were found only in the root (Tracy et al. 2008). Also in experiments with rice and quince protoplasts, different [Ca2+]cyt changes were induced under sodium stress and under osmotic stress (Kader et al. 2007, D'Onofrio and Lindberg 2009).
A proposed model for calcium and pH-signaling under salinity stress is reviewed in Kader and Lindberg (2008, 2010).
7. Aluminium and heavy metal stress signaling in plants
7.1 Aluminium toxicity to plants
Aluminium (Al) toxicity in plants is a serious factor limiting crop production in acidic soils, affecting up to 40% of the world's arable soils (Haug 1984, Foy 1984). When the soil pH decreases below 5, Al3+ is solved in the soil, causing harmful effects on plant roots. At pH 4 the dominating species is Al(H2O)3+; at higher pH also Al(OH)2+, Al(OH)2+, Al(OH)30 and Al(OH)4- are present, besides sulphate complexes and polynuclear species (Lindsay 1979).When roots are subjected to Al they become stunted and damaged and root hair development is poor (Clarkson 1965). The uptake of water and minerals is severely inhibited.
At a low pH Al mainly binds to the root apex and inhibits root elongation (Ryan, DiTomaso and Kochian 1993, Kochian 1995, Matsomoto 2000, Barcelo and Poschenrieder 2002). Aluminium affects the transmembrane potential of root cells and inhibits ATPase activities. After cultivation of sugar beets in the presence of low pH and/or AlCl3, the transmembrane potential, PD, between the vacuole and external medium, PDv, of root cells was largely depolarized (Lindberg, Szynkier and Greger 1991). Since the effect of dinitrophenol was negligable, it was suggested that Al interacts with the active component of the PD. This was confirmed by experiments showing that Al inhibits the plasma membrane ATPase activity (Lindberg and Griffiths 1993) as well as proton transport (Matsumoto 1988). Lipid analysis of sugar beet plasma membranes showed that Al treatment during cultivation caused an increase in the ratio of phoshatidylcholine: phosphatidylethanolamine (Lindberg and Griffiths 1993). The lipid changes correlated with the observed change in the Km for the MgATPase, and, therefore, it was concluded that Al could bind to the membrane-bound enzyme and/or modify the lipid environment. The inhibition of the ATPase activity causes a reduced uptake of minerals. In the soil, phosphate can precipitate with Al and cause phosphate deficiency in plants (Horst, Wager and Marschner 1982)
The in vivo and in vitro effects of Al differ. Addition of low concentrations of Al (10-50 æM) to plant roots cultivated without Al caused a fast hyperpolarization of PDv, and of PDc, the membrane potential across the plasma membrane. A depolarization of PDc was only obtained at pH 6.5 (Lindberg, Szynkier and Greger 1991). At the latter pH the dominant species is Al(OH)30 , which is uncharged and can easily penetrate membranes. It was shown using artificial liposome vesicles that Al uptake was facilitated at neutral pH, compared with pH 4 and 5 (Shi and Haug 1988). Therefore, toxic effects of Al on plants can occur also at a neutral pH.
Most of the Al binds to the cell walls of root epidermal and cortical cells (Delhaize, Ryan and Randall 1993) and to the plasma membranes, but it can also penetrate the plasma membrane (Lazof et al. 1994). When Al enters into a cell cytosol it can inhibit cell division in the meristem, and cell elongation in the elongation zone (Baluska, Parker and Barlow 1993) probably by binding to nucleic acids (Matsumoto et al. 1976).
7.2 Aluminium interferes with calcium homeostasis
A disruption of cytosolic calcium homeostasis is a primary trigger of Al toxicity. Calcium plays an important role in cell division and cell expansion. For instance, transient changes in the cytosolic calcium concentration, [Ca2+cyt], have been observed to accompany the mitotic mechanism (Hepler 1994). Calcium promotes elongation in many plant cells (Takahashi, Scott and Suge 1992, Levina et al. 1995) and calcium antagonists can block elongation (Cho and Hong 1995). Expansion of tip-growing plant cells, such as pollen tubes, is depending on sustained gradients in Ca2+cyt (Clarkson, Brownlee and Ayling 1988, Felle and Hepler 1997, Wymer, Bibikova and Gilroy 1997). A maintained homeostatic control is, therefore, necessary for cell viability (Bush 1995).
Aluminium affects the calcium homeostasis in a cell by inhibition of calcium uptake or efflux (Lindberg 1990). In 1-h experiments with intact sugar beet plants both the metabolic influx of 45Ca2+ and K+(86Rb+), and the efflux of 45Ca2+ were inhibited in the presence of Al (Lindberg 1990). Aluminium at a low concentration and low pH can elevate the [Ca2+cyt] (Lindberg and Strid 1997). Aluminium may also interact with the phosphoinositide signaling pathway. Both AlCl3 and Al-citrate inhibited the phospholipid C (PLC) action in a dose-dependent manner (Jones and Kochian 1995).