胞质Ca2+参与外源H2S促进盐碱胁迫下裸燕麦种子萌发

硫化氢（Hydrogen sulfide，H₂S）是植物新型气体信号分子，钙离子（Calcium，Ca²⁺）为重要的第二信使，两者在植物逆境响应中分别发挥着重要作用。为明确胞质Ca²⁺在外源H₂S促进盐碱胁迫下作物种子萌发中的作用，以裸燕麦（Avena nude）为材料，采用培养皿培养，以混合盐碱（NaCl、Na₂SO₄、Na₂CO₃、NaHCO₃的摩尔比为12：8：1：9）模拟甘肃裸燕麦种植地盐碱环境，蒸馏水为对照，测定了胞外Ca²⁺螯合剂乙二醇-双-（2-氨基乙醚）四乙酸（EGTA）、质膜Ca²⁺通道阻断剂氯化镧（LaCl₃）、液泡Ca²⁺释放抑制剂钌红（RR）和内质网钙泵阻断剂毒胡萝卜素（Thaps）分别与H₂S供体硫氢化钠（NaHS）共处理下种子的发芽势、发芽率、发芽指数、活力指数、平均发芽速率、胚根长和胚芽长7个发芽指标，利用隶属函数分析方法综合评价胞质Ca²⁺对H₂S缓解盐碱胁迫抑制种子萌发的影响。结果表明，随着盐碱胁迫浓度增大，裸燕麦种子的发芽势、发芽率、发芽指数、活力指数、平均发芽速率、胚根长和胚芽长显著下降。与对照相比，15~75 mmol·L^-1盐碱胁迫导致裸燕麦种子萌发的隶属函数综合评价值（D）显著降低，30 mmol·L^-1盐碱胁迫下D值下降了73.1%；100~1 000 μmol·L^-1 NaHS不同程度提高了裸燕麦种子萌发的D值，且100 μmol·L^-1 NaHS缓解30 mmol·L^-1盐碱胁迫下D值下降的作用最大；EGTA、LaCl₃和RR均显著逆转了100 μmol·L^-1 NaHS对30 mmol·L^-1盐碱胁迫下D值下降的缓解作用，而Thaps对NaHS的作用无显著影响。表明胞质Ca²⁺参与外源H₂S促进盐碱胁迫下裸燕麦种子萌发的信号传导过程，且胞质Ca²⁺主要来源于胞外Ca²⁺的内流和液泡中Ca²⁺的释放。

Cytosolic Ca2+ Involved in Seed Germination of Naked Oat Enhanced by Exogenous H2S under Saline-Alkali Stress

Hydrogen sulfide(H₂S) is a new gas signal molecule and calcium(Ca²⁺) is an important second messenger. Both play important roles in plant adversity response, respectively. The experiment was conducted to clarify the role of cytosolic Ca²⁺ in germination of crop seed promoted by exogenous H₂S under saline-alkali stress. Naked oat(Avena nude) was selected as experimental material which was cultured in petri dishes in incubator. Mixed saline-alkaline solution(NaCl:Na₂SO₄:Na₂CO₃:NaHCO₃ with the mole ratio of 12:8:1:9) was used to simulate the saline-alkaline stress of naked oat growing areas in Gansu Province, extracellular Ca²⁺ chelator ethylene glycol-bis-(2-aminoethylether)-N, N, N', N'-tetraacetic acid(EGTA), plasma membrane Ca²⁺ channel blocker lanthanum chloride(LaCl₃), tonoplast Ca²⁺ release inhibitor ruthenium red(RR), and endoplasmic reticulum calcium pump blocker thapsigargin(Thaps) were combined with sodium hydrosulfide(NaHS, a donor of H₂S), respectively, and distilled water treatments were set as control. Seven germination indexes including germination potential, germination rate, germination index, vigor index, average germination speeds, radicle length, and plantule length of naked oat under control, saline-alkaline stress and co-treatments were determined. The membership function analysis was performed to comprehensively evaluate the effect of cytosolic Ca²⁺ on seed germination of naked oat enhanced by H₂S under saline-alkali stress. The germination potential, germination rate, germination index, vigor index, average germination rate, radicle length and plantule length of naked oat seeds decreased significantly with the increase of saline-alkaline concentration. Compared with the control, the 15-75 mmol·L^-1 salt-alkaline stresses resulted in a significant decrease in the membership function comprehensive evaluation value D, D value decreased by 73.1% under 30 mmol·L^-1 saline-alkali stress. The 100-1 000 μmol·L^-1 NaHS increased the D value to varying degrees, and 100 μmol·L^-1 NaHS had the most significant mitigation effect on the decrease of D value under 30 mmol·L^-1 salt-alkaline stress. This mitigation effect of NaHS was weakened by adding EGTA, LaCl₃ and RR, respectively; however, Thaps had no significant effect on the alleviating effect of NaHS to the decrease of D value under 30 mmol·L^-1 salt-alkaline stress. These results show that Ca²⁺ involved signal transduction in promoting of naked oat seed germination induced by H₂S under saline-alkali stress, and cytosolic Ca²⁺ mainly comes from the influx of extracellular Ca²⁺ and the release of Ca²⁺ in vacuoles.