Biochemical characterization of plasma membrane H+-ATPase activation in guard cell protoplasts of Arabidopsis thaliana in response to blue light.
Biochemical characterization of plasma membrane H+-ATPase activation in guard cell protoplasts of Arabidopsis thaliana in response to blue light.
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DOI：
10.1093/pcp/pci104
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发表时间：
2005-06
影响因子：
4.9
通讯作者：
Kumi Ueno;T. Kinoshita;Shin-Ichiro Inoue;T. Emi;K. Shimazaki
中科院分区：
文献类型：
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作者：
Kumi Ueno;T. Kinoshita;Shin-Ichiro Inoue;T. Emi;K. Shimazaki
最近的遗传分析表明，向光素(Phot1和Pho2)在拟南芥气孔开放过程中作为蓝光受体发挥作用，但没有提供这方面的生化证据。我们从拟南芥中制备了大量的保卫细胞原生质体。免疫学方法表明，野生型植物和光合作用突变体的保卫细胞原生质体中存在光敏素1，野生型植物和光敏1突变体的保卫细胞原生质体中存在光敏2，而光敏1和光敏2双突变体的保卫细胞原生质体中既不存在光敏1，也不存在光敏2。然而，所有这些植物的质膜H+-ATPase的含量都是相同的。蓝光可诱导野生型保卫细胞原生质体、光敏素的单一突变体(Pho1-5和Pho2-1)和玉米黄质缺失突变体(npq1-2)的原生质体产生H+泵，但对Pho1、Pho2双突变体的H+泵作用不明显。此外，野生型保卫细胞原生质体对蓝光的响应增加了ATP的水解率和14-3-3蛋白与H+-ATPase的结合。这些结果表明，PHY1和PHY2介导了质膜H+-ATPase在蓝光下的激活，说明拟南芥保卫细胞原生质体可用于气孔功能的生化分析。我们测定了质膜H+-ATPase的同源基因，发现在保卫细胞原生质体中表达了所有功能质膜H+-ATPase的同源基因(AHA1-11)。
Recent genetic analysis showed that phototropins (phot1 and phot2) function as blue light receptors in stomatal opening of Arabidopsis thaliana, but no biochemical evidence was provided for this. We prepared a large quantity of guard cell protoplasts from Arabidopsis. The immunological method indicated that phot1 was present in guard cell protoplasts from the wild-type plant and the phot2 mutant, that phot2 was present in those from the wild-type plant and the phot1 mutant, and that neither phot1 nor phot2 was present in those from the phot1 phot2 double mutant. However, the same amounts of plasma membrane H+-ATPase were found in all of these plants. H+ pumping was induced by blue light in isolated guard cell protoplasts from the wild type, from the single mutants of phototropins (phot1-5 and phot2-1), and from the zeaxanthin-less mutant (npq1-2), but not from the phot1 phot2 double mutant. Moreover, increased ATP hydrolysis and the binding of 14-3-3 protein to the H+-ATPase were found in response to blue light in guard cell protoplasts from the wild type, but not from the phot1 phot2 double mutant. These results indicate that phot1 and phot2 mediate blue light-dependent activation of the plasma membrane H+-ATPase and illustrate that Arabidopsis guard cell protoplasts can be useful for biochemical analysis of stomatal functions. We determined isogenes of the plasma membrane H+-ATPase and found the expression of all isogenes of functional plasma membrane H+-ATPases (AHA1-11) in guard cell protoplasts.