Extracellular Nucleotides and Apyrases Regulate Stomatal Aperture in Arabidopsis
Extracellular Nucleotides and Apyrases Regulate Stomatal Aperture in Arabidopsis
复制标题
DOI：
10.1104/pp.111.174466
复制
发表时间：
2011-08-01
期刊：
影响因子：
7.4
通讯作者：
Roux, Stanley J.
中科院分区：
文献类型：
--
作者：
Clark, Greg;Fraley, Devin;Roux, Stanley J.
本研究探讨了胞外核苷酸和apyrase酶在调节气孔开度中的作用。以往的研究表明，APY1和APY2两种apyrase在拟南芥中的表达与细胞生长和分泌活性密切相关。通过逆转录-聚合酶链式反应和免疫印迹分析，这两种基因在保卫细胞原生质体中都有强烈的表达。APY1和APY2启动子活性分析表明，这两个apyrase的表达与有利于气孔开放的条件有关。相应地，免疫印迹数据表明，当保卫细胞从黑暗进入光明时，这些细胞原生质体中APY的表达迅速上升。多克隆抗体对胞外APYrase活性的短期抑制和对APY1和APY2转录水平的长期抑制都显著扰乱了正常的光下气孔行为。气孔开度对腺苷5‘-[γ-硫代]三磷酸或腺苷5’-[β-硫代]二磷酸的处理表现出双相反应，低浓度诱导气孔打开，高浓度诱导关闭。等量浓度的腺苷5‘-O-硫代磷酸对孔径无影响。两种哺乳动物嘌呤受体抑制剂可阻断三磷酸腺苷、S和5‘-[β-硫代]二磷酸诱导的气孔开放和关闭，也部分阻断了脱落酸诱导气孔关闭和光诱导气孔开放的能力。用三磷酸腺苷-伽马-S处理表皮可诱导一氧化氮和活性氧水平升高，并从基因上抑制这些物质的合成，从而阻断核苷酸对气孔开度的影响。荧光素酶分析表明，诱导气孔关闭或开放的处理也诱导保卫细胞释放ATP。这些数据支持这一新的结论，即胞外核糖核酸酶和胞外核苷酸在调节气孔功能方面起着关键作用。
This study investigates the role of extracellular nucleotides and apyrase enzymes in regulating stomatal aperture. Prior data indicate that the expression of two apyrases in Arabidopsis (Arabidopsis thaliana), APY1 and APY2, is strongly correlated with cell growth and secretory activity. Both are expressed strongly in guard cell protoplasts, as determined by reverse transcription-polymerase chain reaction and immunoblot analyses. Promoter activity assays for APY1 and APY2 show that expression of both apyrases correlates with conditions that favor stomatal opening. Correspondingly, immunoblot data indicate that APY expression in guard cell protoplasts rises quickly when these cells are moved from darkness into light. Both short-term inhibition of ectoapyrase activity by polyclonal antibodies and long-term suppression of APY1 and APY2 transcript levels significantly disrupt normal stomatal behavior in light. Stomatal aperture shows a biphasic response to applied adenosine 5'-[gamma-thio]triphosphate (ATP gamma S) or adenosine 5'-[beta-thio] diphosphate, with lower concentrations inducing stomatal opening and higher concentrations inducing closure. Equivalent concentrations of adenosine 5'-O-thiomonophosphate have no effect on aperture. Two mammalian purinoceptor inhibitors block ATP gamma S- and adenosine 5'-[beta-thio] diphosphate-induced opening and closing and also partially block the ability of abscisic acid to induce stomatal closure and of light to induce stomatal opening. Treatment of epidermal peels with ATP gamma S induces increased levels of nitric oxide and reactive oxygen species, and genetically suppressing the synthesis of these agents blocks the effects of nucleotides on stomatal aperture. A luciferase assay indicates that treatments that induce either the closing or opening of stomates also induce the release of ATP from guard cells. These data favor the novel conclusion that ectoapyrases and extracellular nucleotides play key roles in regulating stomatal functions.