Luo Hao, Lin Xin, Li Ling, Lin Lingxiao, Zhang Chao, Lin Senjie
State Key Laboratory of Marine Environmental Science, Department of Marine Biological Sciences and Technology, College of Ocean and Earth Sciences, Xiamen University, Xiamen 361102, China.
Department of Biochemistry, Province Key Laboratory of Biochip, School of Basic Medical Science and Institute of Genetic Engineering, Southern Medical University, Guangzhou, China.
Environ Microbiol. 2017 Nov;19(11):4506-4518. doi: 10.1111/1462-2920.13899. Epub 2017 Sep 15.
The ability to utilize dissolved organic phosphorus (DOP) is important for phytoplankton to survive the scarcity of dissolved inorganic phosphorus (DIP), and alkaline phosphatase (AP) has been the major research focus as a facilitating mechanism. Here, we employed a unique molecular ecological approach and conducted a broader search for underpinning molecular mechanisms of adenosine triphosphate (ATP) utilisation. Cultures of the dinoflagellate Karenia mikimotoi were set up in L1 medium (+P), DIP-depleted L1 medium (-P) and ATP-replacing-DIP medium (ATP). Differential gene expression was profiled for ATP and +P cultures using suppression subtractive hybridisation (SSH) followed by 454 pyrosequencing, and RT-qPCR methods. We found that ATP supported a similar growth rate and cell yield as L1 medium and observed DIP release from ATP into the medium, suggesting that K. mikimotoi cells were expressing extracellular hydrolases to hydrolyse ATP. However, our SSH, qPCR and enzymatic activity assays indicated that 5'-nucleotidase (5NT), rather than AP, was responsible for ATP hydrolysis. Further gene expression analyses uncovered that intercellular purine metabolism was significantly changed following the utilisation of ATP. Our findings reveal a multi-faceted machinery regulating ATP utilisation and P metabolism in K. mikimotoi, and underscore AP activity is not the exclusive indicator of DOP utilisation.
利用溶解有机磷（DOP）的能力对于浮游植物在溶解无机磷（DIP）稀缺的情况下生存至关重要，而碱性磷酸酶（AP）作为一种促进机制一直是主要的研究重点。在此，我们采用了一种独特的分子生态学方法，对三磷酸腺苷（ATP）利用的潜在分子机制进行了更广泛的探索。在L1培养基（+P）、DIP耗尽的L1培养基（-P）和ATP替代DIP培养基（ATP）中建立了米氏凯伦藻的培养物。使用抑制性消减杂交（SSH）结合454焦磷酸测序和RT-qPCR方法，对ATP和+P培养物进行了差异基因表达分析。我们发现ATP支持的生长速率和细胞产量与L1培养基相似，并观察到ATP向培养基中释放DIP，这表明米氏凯伦藻细胞表达了细胞外水解酶来水解ATP。然而，我们的SSH、qPCR和酶活性测定表明，负责ATP水解的是5'-核苷酸酶（5NT），而不是AP。进一步的基因表达分析发现，在ATP利用后，细胞内嘌呤代谢发生了显著变化。我们的研究结果揭示了米氏凯伦藻中调节ATP利用和磷代谢的多方面机制，并强调AP活性不是DOP利用的唯一指标。
