Departamento de Biología Vegetal, Avda. Diagonal n° 643, Facultad de Biología, Universidad de Barcelona, 08028 Barcelona, Spain.
Plant Physiol Biochem. 2013 Feb;63:1-7. doi: 10.1016/j.plaphy.2012.11.008. Epub 2012 Nov 23.
Phytochrome (phy) signalling in plants may be transduced through protein phosphorylation. Mitogen-activated protein kinase (MAP-kinase, MAPK) activity and the effect of R (red) and FR (far-red) light irradiation on MAPK activity were studied in etiolated Cucumis sativus L. cotyledons. By in vitro protein phosphorylation and in-gel assays with myelin basic protein (MBP), a protein band (between 48 and 45 kDa) with MAPK-like activity was detected. The addition to the phosphorylation buffer of specific protein phosphatase (PTP) inhibitors (Na(3)VO(4) and NaF) and genistein, apigenin or PD98059 as MAPK inhibitors allowed us to confirm the MAPK activity of the protein band. Irradiation of etiolated cotyledons with FR light for 5, 10 or 60 min rapidly and transiently stimulated the MAPK activity of the protein band. This suggests that there was a very low fluence response (VLFR) of phys. In addition, 15 min of R light irradiation or a sequential treatment of 15 min of R plus 5 min of FR also increased MAPK activity. The stimulatory effect of R light was also attributed to the same photoreceptor, which suggests that MAPKs are involved in phytochrome signal transduction. Protein immunoprecipitation and immunoblotting analysis with the polyclonal antibody anti-pERK1/2 (Tyr 204) and the monoclonal antibody anti-phosphotyrosine PY20 allowed us to recognize the above mentioned protein band as two proteins with molecular masses (M(r)) of approximately 47 and 45 kDa, and MAPK activity. The biochemical and immunological properties showed by the proteins detected indicated that they were members of the MAPK family phosphorylated in tyrosine residues.
植物中的光敏素（phy）信号可能通过蛋白质磷酸化传递。研究了拟南芥黄化子叶中丝裂原激活蛋白激酶（MAPK，MAPK）的活性以及 R（红）和 FR（远红）光照射对 MAPK 活性的影响。通过体外蛋白磷酸化和用髓鞘碱性蛋白（MBP）进行的凝胶内分析，检测到具有 MAPK 样活性的蛋白带（在 48 和 45 kDa 之间）。在磷酸化缓冲液中加入特定的蛋白磷酸酶（PTP）抑制剂（Na3VO4和 NaF）和作为 MAPK 抑制剂的金雀异黄素、芹菜素或 PD98059，可以确认蛋白带的 MAPK 活性。用 FR 光照射黄化子叶 5、10 或 60 min 可快速且短暂地刺激蛋白带的 MAPK 活性。这表明存在非常低的光通量反应（VLFR）。此外，15 min 的 R 光照射或 15 min 的 R 加 5 min 的 FR 的连续处理也增加了 MAPK 活性。R 光的刺激作用也归因于相同的光受体，这表明 MAPKs 参与了光敏素信号转导。用多克隆抗体抗-pERK1/2（Tyr 204）和单克隆抗体抗磷酸酪氨酸 PY20 进行蛋白免疫沉淀和免疫印迹分析，使我们能够识别上述蛋白带为两种分子量（Mr）约为 47 和 45 kDa 的蛋白，以及 MAPK 活性。所检测到的蛋白的生化和免疫学特性表明它们是磷酸化酪氨酸残基的 MAPK 家族成员。
