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星星草(Puccinellia tenuiflora)叶绿体Na_2CO_3胁迫应答的生理学与定量蛋白质组学研究
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摘要
盐碱胁迫是严重影响植物生长,限制产量和区域分布的重要因素之一。盐碱胁迫会引起植物水分缺失,破坏渗透平衡和氧化平衡,导致叶片气孔导度降低,抑制光合作用,对植物的光能利用和CO2同化作用造成影响。星星草(Puccinellia tenuiflora)作为一种耐盐碱的禾本科植物,能够在pH9-10的环境中完成生活史,是研究植物盐碱耐受机制的理想模式植物。
     本研究以星星草叶绿体为材料,利用形态学、生理学与定量蛋白质组学研究策略对不同Na2CO3胁迫条件下(150mM-12h,200mM-12h,150mM-24h和200mM-24h)的胁迫应答机制进行了研究。我们发现,Na2C03胁迫一定程度破坏了叶绿体结构,抑制了光反应活性和CO2同化过程,限制了星星草的生长。Na+的大量积累对星星草产生了离子毒害,并且破坏了渗透平衡;而K+/Na+比值、Ca2+和Mg2+含量的降低抑制了多种酶的活性,对植物的生长产生影响。丙二醛和电解质外渗率的提高也证实星星草膜结构受到损伤,而脯氨酸和可溶性糖的大量积累有利于保护膜结构的完整性。抗氧化酶系统的变化提高了植株/叶绿体对氧化胁迫的防御能力。定量蛋白质组学结果揭示了叶绿体中68种Na2CO3应答蛋白质,它们主要参与捕光复合体、光系统Ⅱ、光合电子传递链和光系统Ⅰ的形成、能量代谢、卡尔文循环、光合色素代谢、基础代谢、胁迫防御、转录、蛋白质合成与命运,以及信号转导等过程。通过对这些结果的分析,我们推测星星草叶绿体可能通过以下几种策略应答Na2CO3胁迫:(1)通过降低叶绿素含量和抑制叶绿素a/b结合蛋白的表达减少过剩光能的吸收;(2)通过增加光化学淬灭和加速叶黄素循环提高热耗散能力,减少过量光能对光合结构的损伤;(3)通过提高超氧化物歧化酶(SOD)和抗坏血酸过氧化物酶(APX)等抗氧化酶的活性及加速叶绿素合成底物和中间产物的分解减轻氧化胁迫损伤,同时利用脯氨酸和可溶性糖的大量积累来缓解渗透胁迫,并清除活性氧类物质(ROS),维持氧化还原平衡。
     总之,我们通过综合运用形态学、生理学和蛋白质组学研究手段,分析了星星草响应Na2CO3胁迫的光合应答机制,为深入研究植物耐盐碱机制提供了新的线索。
Alkali-salinity is one of the most significant abiotic stresses and it limits the productivity and geographical distribution of plants. Alkali-salinity stress imposed severe effects on plant by causing water deficit, which subsequently reduce the stomatal conductance, disturbances in osmotic and oxidative homeostasis. Photosynthesis is inhibited, such as the recuction of light energy utility and CO2assimilation in plants.
     Puccinellia tenuiflora is a halophytic species belonging to the Gramineae, and capable of completing its life cycle under salt environment with pH9-10. Therefore, P. tenuiflora is considered as an ideal model plant for studying the salt-tolerant mechanisms in plants. In this study, the Na2CCO3responsive molecular mechanisms in P. tenuiflora chloroplast was investigated using a combined physiological and proteomic approach., we found the chloroplast structure was affected, and light reaction acitivity and CO2assimilation were inhibited Under various Na2CO3treatments (150mM-12h,200mM-12h,150mM-24h, and200mM-24h). These led to the growth of P. tenuiflora seedling reduced. Accumulation of Na+causes ion-specifis stress and dissipates the membrane potential. Altered K+/Na+ration and reduced Ca2+and Mg2+contents can inhibit the activity of many essential enzymes and metabolism pathways, which finally lead to growth inhibition. The remarkable increased result of malondialdehyde (MDA) and relative electrolyte leakage further illustrated the increase of membrane lipid peroxidation and membrane permeability. Moreover, the accumulation of proline and soluble sugar enhance the ability of osmotic regulation are helpful to preserve the integrity of cell structure. In addition, dynamic changes of antioxidant system play important roles in the protection of plant/chloroplast response to Na2CO3. Furthermore, using an isobaric tags for relative and absolute quantitation (iTRAQ) approach,68differentially expressed proteins were identified in P. tenuiflora chloroplast in response to Na2CO3. These proteins were mainly involved in the formation of light harvest complex, photosystem Ⅱ, photosynthetic electron transfer chain, and photosystem Ⅰ, energy metabolism, Calvin cycle, photosynthetic pigment metabolism, metabolism, stress and defence, transcription, protein synthesis and fate, as well as signaling transduction.
     Taken together, our physiological and proteomics results reveal that P. tenuiflora chloroplast may utilize various strategies to cope with Na2CO3treatment. They include:(1) reduce excessive light energy absorption by decreasing chlorophyll content and inhibiting chlorophyll a/b binding protein expression;(2) enhance the heat dissipation by increasing photochemical quenching and accelerating xanthophyll cycle, and thereby reduce the damage of photosynthetic apparatus caused by excessive light energy;(3) minimize the oxidative injury by elevating the specific antioxidant system and enhancing the dissolution of xanthophyll synthetic substrates and intermediate products. In addition, the considerable accumulation of proline and soluble sugar can alleviate osmotic stress, as well as scavenge reactive oxygen species (ROS) to maintain oxidative balance.
     This study lays a solid foundation for further research on salt-tolerant mechanisms in the Gramineae.
引文
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