Activation of plastid-localized latent polyphenol oxidase by autophagy induced by sucrose-starvation

多酚氧化酶(PPO)，在细胞质中被翻译为前体蛋白，然后以潜伏期的形式运输和定位在质体中。然而，PPO的底物定位于另一个隔室(液泡)。植物多酚是众所周知的最受欢迎的酶之一，与受伤或患病组织的褐变有关。然而，它在活的植物细胞中的生物学功能尚不清楚。在目前的研究中，我们证明了胞质定位的潜伏PPO很可能是通过参与自噬的蛋白水解性裂解来激活的。主要实验结果如下：1)从甘薯细胞中克隆了2个PPO亚型的cDNA。2)细胞转移到新鲜培养基、细胞增殖和茉莉酸甲酯处理诱导了PPO的表达。3)蔗糖饥饿或茉莉酸甲酯对细胞中潜伏的PPO进行了蛋白分解激活。4)部分纯化了液泡定位的半胱氨酸内肽酶(PPOase)，该酶可以裂解潜伏的PPO。PPOase对PPO具有较高的底物专一性。5)E-或3-甲基腺嘌呤能明显抑制去蔗糖培养的甘薯细胞中潜伏的PPO的蛋白分解。蔗糖饥饿使叶绿体数量显著减少，但E-和3-甲基腺嘌呤均能抑制叶绿体的降解。6)从E-处理的细胞中分离到空泡。在荧光显微镜下观察到含有许多DAPI染色结构的液泡，这些实验结果表明甘薯细胞的自噬是由蔗糖饥饿诱导的，活性的PPO是由液泡中参与半胱氨酸内肽酶的胞体定位的潜在PPO的蛋白水解性裂解产生的。

Polyphenol oxidase (PPO) that is translated as precursor protein in cytoplasm, then transported and localized in plastids as a latent form. However, PPO's substrates are localized in the other compartment (vacuoles). Plant PPO is well known as one of the most popular enzymes that are involved in browning of the injured or diseased tissues. However, its biological function in the living plant cell is not clear. In the present study, we demonstrate that the plastid-localized latent PPO is most probably activated by proteolytic cleavage involved in autophagy. Main experimental results are shown as follows.1) We cloned 2 PPO isoform cDNAs from sweet potato cells.2) Expression of PPO was induced by cell transfer to fresh medium, cell proliferation and treatment with methyl jasmonate.3) Latent PPO was activated by proteolytic cleavage in the cells by sucrose starvation or methyl jasmonate.4) A vacuole-localized cysteine endopeptidase (PPOase) that can cleave the latent PPO was partially purified. PPOase showed a high substrate specificity for PPO.5) Proteolytic cleavage of latent PPO in the sweet potato cells that had been cultured in sucrose-deleted medium was markedly inhibited by E-64 or 3-methyladenine. Plastid number severely decrease by sucrose starvation, but both E-64 and 3-methyladenine also inhibited the breakdown of plastids.6) Vacuoles were isolated from E-64 treated cells that had been cultured in sucrose-deleted medium. The vacuoles containing many DAPI-stained structures that seemed to be plastids were observed under fluorescent microscopy.These experimental results indicate that autophagy is induced by sucrose starvation in sweet potato cells, and the active PPO is generated by proteolytic cleavage of the plastid-localized latent PPO involved in cysteine endopeptidase in the vacuoles.