Lorenzo O, Rodríguez D, Nicolás G, Rodríguez P L, Nicolás C
Departamento de Fisiología Vegetal, Facultad de Biología, Universidad de Salamanca, Plaza de los Doctores de la Reina s/n. 37007-Salamanca, Spain.
Plant Physiol. 2001 Apr;125(4):1949-56. doi: 10.1104/pp.125.4.1949.
An abscisic acid (ABA)-induced cDNA fragment encoding a putative protein phosphatase 2C (PP2C) was obtained by means of differential reverse transcriptase-polymerase chain reaction approach. The full-length clone was isolated from a cDNA library constructed using mRNA from ABA-treated beechnut (Fagus sylvatica) seeds. This clone presents all the features of plant type PP2C and exhibits homology to members of this family such as AthPP2CA (61%), ABI1 (48%), or ABI2 (47%), therefore it was named FsPP2C1. The expression of FsPP2C1 is detected in dormant seeds and increases after ABA treatment, when seeds are maintained dormant, but it decreases and tends to disappear when dormancy is being released by stratification or under gibberellic acid treatment. Moreover, drought stress seems to have no effect on FsPP2C1 transcript accumulation. The FsPP2C1 transcript expression is tissue specific and was found to accumulate in ABA-treated seeds rather than in other ABA-treated vegetative tissues examined. These results suggest that the corresponding protein could be related to ABA-induced seed dormancy. By expressing FsPP2C1 in Escherichia coli as a histidine tag fusion protein, we have obtained direct biochemical evidence supporting Mg2+-dependent phosphatase activity of this protein.
通过差异逆转录聚合酶链反应方法获得了一个脱落酸（ABA）诱导的编码假定蛋白磷酸酶2C（PP2C）的cDNA片段。从使用ABA处理过的欧洲山毛榉种子的mRNA构建的cDNA文库中分离出全长克隆。该克隆具有植物型PP2C的所有特征，并与该家族成员如AthPP2CA（61%）、ABI1（48%）或ABI2（47%）具有同源性，因此将其命名为FsPP2C1。在休眠种子中检测到FsPP2C1的表达，在ABA处理后种子保持休眠时其表达增加，但在通过层积处理或赤霉素处理解除休眠时其表达下降并趋于消失。此外，干旱胁迫似乎对FsPP2C1转录本积累没有影响。FsPP2C1转录本的表达具有组织特异性，发现在ABA处理的种子中积累，而不是在所检测的其他ABA处理的营养组织中积累。这些结果表明相应的蛋白质可能与ABA诱导的种子休眠有关。通过在大肠杆菌中表达作为组氨酸标签融合蛋白的FsPP2C1，我们获得了支持该蛋白具有Mg2+依赖性磷酸酶活性的直接生化证据。
