Astorquiza Paula Luján, Usorach Javier, Racagni Graciela, Villasuso Ana Laura
Química Biológica, FCEFQN, Universidad Nacional de Río Cuarto, X5804BYA Río Cuarto, Córdoba, Argentina.
Química Biológica, FCEFQN, Universidad Nacional de Río Cuarto, X5804BYA Río Cuarto, Córdoba, Argentina.
Plant Physiol Biochem. 2016 Apr;101:88-95. doi: 10.1016/j.plaphy.2016.01.012. Epub 2016 Jan 27.
The aleurona cell is a model that allows the study of the antagonistic effect of gibberellic acid (GA) and abscisic acid (ABA). Previous results of our laboratory demonstrated the involvement of phospholipids during the response to ABA and GA. ABA modulates the levels of diacylglycerol, phosphatidic acid and diacylglycerol pyrophosphate (DAG, PA, DGPP) through the activities of phosphatidate phosphatases, phospholipase D, diacylglycerol kinase and phosphatidate kinase (PAP, PLD, DGK and PAK). PA and DGPP are key phospholipids in the response to ABA, since both are capable of modifying the hydrolitic activity of the aleurona. Nevertheless, little is known about the mechanism of action of these phospholipids during the ABA signal. DGPP is an anionic phospholipid with a pyrophosphate group attached to diacylglycerol. The ionization of the pyrophosphate group may be important to allow electrostatic interactions between DGPP and proteins. To understand how DGPP mediates cell functions in barley aleurone, we used a DGPP affinity membrane assay to isolate DGPP-binding proteins from Hordeum vulgare, followed by mass spectrometric sequencing. A cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was identified for being bound to DGPP. To validate our method, the relatively abundant GAPDH was characterized with respect to its lipid-binding properties, by fat western blot. GAPDH antibody interacts with proteins that only bind to DGPP and PA. We also observed that ABA treatment increased GAPDH abundance and enzyme activity. The presence of phospholipids during GAPDH reaction modulated the GAPDH activity in ABA treated aleurone. These data suggest that DGPP binds to GAPDH and this DGPP and GAPDH interaction provides new evidences in the study of DGPP-mediated ABA responses in barley aleurone.
糊粉层细胞是一种可用于研究赤霉素（GA）和脱落酸（ABA）拮抗作用的模型。我们实验室之前的研究结果表明，磷脂参与了对ABA和GA的响应过程。ABA通过磷脂酸磷酸酶、磷脂酶D、二酰基甘油激酶和磷脂酸激酶（PAP、PLD、DGK和PAK）的活性来调节二酰基甘油、磷脂酸和二酰基甘油焦磷酸（DAG、PA、DGPP）的水平。PA和DGPP是对ABA响应中的关键磷脂，因为它们都能够改变糊粉层的水解活性。然而，关于这些磷脂在ABA信号传导过程中的作用机制，我们了解得还很少。DGPP是一种阴离子磷脂，其焦磷酸基团连接在二酰基甘油上。焦磷酸基团的离子化对于DGPP与蛋白质之间的静电相互作用可能很重要。为了了解DGPP如何介导大麦糊粉层中的细胞功能，我们使用DGPP亲和膜分析法从大麦中分离出与DGPP结合的蛋白质，随后进行质谱测序。我们鉴定出一种胞质甘油醛-3-磷酸脱氢酶（GAPDH，EC 1.2.1.12）与DGPP结合。为了验证我们的方法，通过脂肪免疫印迹法对相对丰富的GAPDH的脂质结合特性进行了表征。GAPDH抗体与仅结合DGPP和PA的蛋白质相互作用。我们还观察到，ABA处理会增加GAPDH的丰度和酶活性。在GAPDH反应过程中磷脂的存在调节了ABA处理的糊粉层中的GAPDH活性。这些数据表明，DGPP与GAPDH结合，并且这种DGPP与GAPDH的相互作用为大麦糊粉层中DGPP介导的ABA响应研究提供了新的证据。
