Effect Of Cytosol Ca～(2+) And Calcium Channel On Rice Hypersensitive Response

A pathosystem of ricedenthomonas oryzae pv. oryzae was used to investigate (l)characteristic dynamics of cytosolic calcium ions ([Ca'+],) in the compatible andincompatible interaction; (2) effect of calcium channel on [Ca'+1.; (3) effect of [Ca'+],and calcium channel in plasma membrane on hypersensitive response and oxidativeburst in incompatible interaction.Rice (Oryza sativa L.) cultivar IR-BBl0 with resistance gene Xa-I0 and cv. IR24withollt Xa-10 were used. The tested strains of Xanthomonas oryzae pv. oryzae werestrain POX99'pBUavrXal0.Fl with avrXa-10 and POX99" without avrXa-!0.Suspension-cultured rice cells were stained with Evans blue at 4 h-24 h after bacterialinoculation at intervals. The result showed hypersensitive necrosis occurred at 6 h afterinoculation in the incompatible interaction, whereas in the compatible interaction celldeath was postponed to l8 h after inoculation.Extracellular pH and O,'-. H₂O₂ burst were measured by 6 h after inoculation. Theresults showed the first peak of H₂O₂ occurred at 0.5 h after inoculation. At l h afterinoculation, the second peak of H₂O₂ and the O,'- peak occurred. Moreover extracellularpH in the incompatible interaction was apparently higher than that in the colltrol 2 hafter inoculation and later. The data suggested that extracellular alkalization andoxidative burst existed at the early stage of the incompatible interaction between riceand XO o. Therefore, it was speculated that the initial response and the related signalregulation pathways during interaction between rice and Xanthomonas oryzae pv.oryzae were determined by 6 h after inoculation.[Ca'+], was measured by calcium-sensitive fluorescent dyes (fura-2). Cytosolicfree Ca2+ showed characteristic oscillation with constallt baseline and repetitive spikeswhich periodicity was 10 s-20 s or so. [Ca'+]. in "resting" or quiescent state was 120nmol/L. When suspension-cultured rice cells were inoculated with an incompatiblebacterium, [Ca'+], reached its maximum value (600 nmol/L) at 10 min after inoculation.Then [Ca'+], subsided slowly to the "resting" level by 30 min after inoculation.The suspension-cultured rice cells were simultaneously treated with bacterium andcalcium chelator (4 mmol/L EGTA) or calcium ionophore (5 pmol/L A23l87). Theincrease in [Ca'+1. was prevented by calcium chelator. The treatment of calciumionophore resulted in increase in [Ca'+l, compared to addition of bacterium only. Theseeffects were consistent with pharmacological test in which calcium chelator inhibitedthe hypersensitive necrosis, extracellular alkalization and H,O, burst, whereas calciamionophore strengthened these reactions. The data suggested that increase in [Ca'+], wasinvolved in signal transduction leading to the hypersensitive response and oxidativeburst during the interaction between rice and Xanthomonas oryzae pv. oryzae.The suspension-cultured rice cells were simultaneously treated with bacterium andcalcium channel blocker, 200 pmol/L LaCl, or veraPamil. The increase in [Ca'+]. wasprevented by both blockers. The effect was consistent with pharmacological test inwhich calcium channel blockers inhibited the hypersensitive necrosis, extracel1uIaralkalization and H,O, burst. Because LaCl, (Lanthanum chloride) is a calcium channelblocker of plasma membrane and verapamil is a voltage-gated calcium channel blocker,the data suggested that the increase in [Ca'+], derived from extracellular Ca'+ whichinflux through voltage-gated calcium channel in plasma membrane.The suspension-cultured rice cells were treated with EGTA, LaCl, and veraPamil,respectively, at 30 min after bacterial inoculation. All the treatment delayed O, - burst.Moreover, there is no increase in [Ca'+], at 30 min after inocuIation. The data suggestedthat O,'- burst was probably relative to the Ca2+ fluxation over plasma membrane.The study suggested that extracellular Ca2+ influxed to cytosol by voltage-gatedcalc...