Seminar in IARI

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Calcium signalling in plants


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There is no greater object of wonder, there is no greater inning of beauty than the ordered series and Linnaeus. : 

There is no greater object of wonder, there is no greater inning of beauty than the ordered series and Linnaeus. organized complexities of life

Signaling is an essential part of communication : 

Signaling is an essential part of communication Primary signaling Secondary signaling Secondary signaling molecules Cyclic nucleotides Phospho-lipids Ca2+ British biologist Sydney Ringer first demonstrated the biological significance of calcium. In 1883 Hundred year later it is Rasmussen elucidated the secondary signaling role of Ca2+

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UNIQUE CALCIUM: It is believed that calcium stores in cells could have originated primarily for the purpose of defence against the ‘calcium holocaust’ (Campbell 1988). Since the intracellular level of Mg2+ ion is already high, a rapid and large (>10-fold) increase is not possible for this ion Ca2+ ion has the maximum affinity for carboxylate oxygen, 0.82% of (Glu + Asp) over(Asn + His + Gln) amino acids. The important thing is that this translates into an excess of 77860 (Glu + Asp) Ca2+ binds to and dissociates from a protein 100-fold or so faster than Mg2+ (Ochiai 1991)

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Mg octahedral- resists external influences. Ca bipyramide or pentagonal. Cross-links mediated by calcium ion are reversible and thus responsive to a change of conditions.

Physiological roles of Calcium : 

Physiological roles of Calcium As essential nutrient As cell cementing material As secondary signaling molecule

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The principal elements in the system of calcium signaling of plant cells are different Ca2+ channels, Ca2+-ATPases, Ca2+/H+ antiporters, Ca2+ -binding and Ca2+ -dependent proteins

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Ca2+ influx takes place by Ca2+ channels and Ca2+ ATPase Ca2+ eflux takes place by InsP3 receptors, Ryanodin receptors

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Calcium signal decoders Group I EF hand proteins: do not undergo conformational change Act in Ca2+ buffering e.g., Parvabilin, Calbindin Group II EF hand proteins: Calmodulins CDPK

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Stimuli Signal Signal coding Signal decoding Gene expression Response

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An Overview of Calcium Regulation in Cells. Cytosolic calcium concentration is lowered by the actions of the ER calcium pump, the plasma membrane calcium pump, sodium-calcium exchangers, and the mitochondria. Calcium concentration increases in the cytosol because of the opening of calcium channels in the plasma membrane and the release of calcium through the lnsP3 or ryanodine receptor channels in the ER membrane.

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Hyperosmotic Stress Elicits a Transient Ca2+cyt In hyperosmotic stress nonselective cation channel in cell membrane opens Increase in cytoplasmic calcium potentiates the ROS production in Mitochondria In animals this process is associated with respiratiory disorders In plant yet to be characterized (Susana et al., 2002)

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After 10 sec the calcium wave was observed in rhizoids of fucus The speed of calcium wave as estimated as 15 µm/s (Goddard et al., 2000) Calcium wave initiates in hyperosmotic condition

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Mitochondria contributes to intracellular sink for calcium Increase in calcium was observed by mitochondrial calcium tracker X-rhod-EF Reactive oxygen species (ROS) production was monitored as oxidation of 5- (and 6-) chloromethyl-2,7-dichlorodihydrofluorescein diacetate (CM-DCFH2-DA)

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Calcium chelater (Br2 BAPTA) prevented the mitochondrial ROS production Increase in cytosolic calcium is necessary step for mitochondrial ROS production (Susana et al., 2002)

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Proposed Signaling Pathway during Hyperosmotic Stress in Fucus. Osmotic change is sensed by an unidentified osmosensor (1), which induces DPI-sensitive ROS production within 10 s (2). H2O2 is produced at the exterior side of the plasma membrane and may be involved in cell wall strengthening. H2O2 also diffuses into the cell, leading to a localized peripheral intracellular increase. Localized external H2O2 production also increases Ca2 channel activity (3). Downstream events include Ca2 wave propagation during the subsequent 10 to 60 sec (4), followed by Ca2 m increase (5) and mitochondrial ROS production (6). FCCP, carbonylcyanide p-trifluoromethoxyphenyl hydrazone.

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Growing Lilly pollen tube showed tip focused [Ca2+]Cyt gradient oscillation during tube growth by having >3000 nM at apex compare to 150-300 nM in sub apical region (Holdway-clarke et al., 1997) This suggest that [Ca2+]Cyt oscillation may regulates actin dynamics to modulate pollen tube growth through action of actin binding protein (Staigner and Blanchoin, 2006)

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Calcium sensitive actin binding protein like Profilin (Snowman, 2002) occur commonly in pollen tubes (Yokota and Shimmer, 2006) When treated with pollen tube growth inhibitor Laturin-B the oscillatory growth stopped. Pollen tube growth occur in actin dependent manner (Lovy-Wheeler et al., 2007)

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Calcium gradient exhibits oscillatory growth of pollen tube apex (Louis et al., 2008) Pollen tube labelled with fura 2-dextran: A-clear Ca gradient B-decrease after 5min C- 10min D-20min E-Control

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Calmodulin is a primary Ca2+ binding protein in cells Involved in the signal relay mechanism of Ca2+ signaling Have four Ca2+ binding sites separated by a 2 nm long helix After ca2+ binding, it under go conformational change And wraps around the protein to which it is destined to activate

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Structure of Ca2+binding protein Calmodulin (left), sites of Ca2+ binding on four loops of the protein (middle), details of single loop harboring calcium ion (right).

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Calmodulin binding proteins in plants are Myosin V, Kinesin, NAD+ kinase, Glutamate decarboxylase Protein kinases, and Ca2+ ATPases, many more are still unidentified.

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Plant defense is nudged by calcium signaling Hyper sensitive reaction is one of the defense mechanism Plants innate immune signaling cascade involve cytosolic calcium rise and nitric oxide generation which ultimately leads to HR (Wendeheune, 2006) Early step in infection is pathogen perception and calcium influx (Lecourieux et al., 2006)

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Nitric oxide production is responsible for success of HR (Zemojtel et al., 2006) In plant Nitrate Reductase is the potential nitric oxide producer but is it the responsible candidate for HR Nitric oxide production? A, NO generation in the absence (white bar) and presence (gray bar) of the NR inhibitor (1 mM) NaN3. No, then what?

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This says may be NOS or similar enzyme in plant have role in HR NOS like activity proved in infected plant extracts B, NO generation in the absence (white bar) and presence (gray bar) of the NOS inhibitor (20 mM) diphenyleneiodonium chloride(DPI).

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This NOS activity is dependent on calcium concentration There is a link between the calcium, Calmodulin and NOS in the plant pathogen response signaling cascade (Ali et al., 2007) C, Effect of the Ca2+ chelator EGTA on NO generation.

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(Wei Ma et al., 2008) D, All assays included 1 mM NaN3. In the presence of this NR inhibitor, NO generation was monitored in the presence and absence of 1 mM Ca2+ and 1 mM CaM. E, In the presence (left) or absence (right) of 1 mM CaM, NO generation was monitored in the absence (white bars) or presence (gray bars) of 200 mM W7. All assays in A to C included 1 mM Ca2+ and 1 mM CaM.

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Co- infiltration of Calmodulin antagonist with pathogen impairs the hyper sensitive reaction(Di fluoperazine dihydrochloride-w7) But Calmodulin antagonist have no effect on the sustained calcium rise The calmodulin antagonist blocks the Nitric Oxide generation NO binding fluorophore of DAF2DA is generated only within cells upon cleavage of the ester linkage; hence, the dye responds only to intracellular NO. NO-specific fluorescent dye 4,5-diaminofluorescein diacetate

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The CaM antagonist W7 prevents HR in wild-type plants. A, Inoculated regions of the leaves are highlighted by boxes. As shown in the top three panels, HR development becomes visible at 13 h after inoculation

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Not only calmodulin.. Calmodulin like proteins are known to involve in HR Silencing the CML gene APR134 suppressed HR (Chiasson et al., 2005) Calmodulin like protein CML 24 involved in controlling the NO accumulation (Tsai et al., 2007) cml24 mutant show impaired HR (Wei Ma et al., 2008)

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Loss-of-function cml24-4 plants show an impaired HR phenotype. A, Inoculated areas of the leaves are highlighted by boxes. In the top two panels, HR development B, Measurement of ion leakage in response to inoculation Hypersensitive reaction was evident in wild type

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CONCLUSION Calcium plays versatile role in various metabolic responses They are - Abiotic stress -Pollen tube growth -Plant defense mechanism etc. Calmodulin and related proteins together are involving in calcium signal relying

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FUTURE PERSPECTIVES As Ca2+ signaling is essential in pollen tube growth, it must have role in self incompatibility and male sterility. Thorough understanding of Ca2+ signaling have a good potential to tackle the problems of biotic and abiotic stresses A new field called kinomics is on its way to decode the role of different kinases in Ca2+ signaling Can't we think of deriving sustainable solution for CO2 dependent global worming by unraveling Ca2+ signaling in CO2 sinking marine flora?

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