Dong Yan-Shan, Fu Chun-Hua, Su Peng, Xu Xiang-Ping, Yuan Jie, Wang Sheng, Zhang Meng, Zhao Chun-Fang, Yu Long-Jiang
Institute of Resource Biology and Biotechnology, Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China.
Key Laboratory of Molecular Biophysics Ministry of Education, Huazhong University of Science and Technology, Wuhan, China.
Physiol Plant. 2016 Jan;156(1):13-28. doi: 10.1111/ppl.12382. Epub 2015 Oct 10.
Browning phenomena are ubiquitous in plant cell cultures that severely hamper scientific research and widespread application of plant cell cultures. Up to now, this problem still has not been well controlled due to the unclear browning mechanisms in plant cell cultures. In this paper, the mechanisms were investigated using two typical materials with severe browning phenomena, Taxus chinensis and Glycyrrhiza inflata cells. Our results illustrated that the browning is attributed to a physiological enzymatic reaction, and phenolic biosynthesis regulated by sugar plays a decisive role in the browning. Furthermore, to confirm the specific compounds which participate in the enzymatic browning reaction, transcriptional profile and metabolites of T. chinensis cells, and UV scanning and high-performance liquid chromatography-mass spectrometry (HPLC-MS) profile of the browning compounds extracted from the brown-turned medium were analyzed, flavonoids derived from phenylpropanoid pathway were found to be the main compounds, and myricetin and quercetin were deduced to be the main substrates of the browning reaction. Inhibition of flavonoid biosynthesis can prevent the browning occurrence, and the browning is effectively controlled via blocking flavonoid biosynthesis by gibberellic acid (GA3 ) as an inhibitor, which further confirms that flavonoids mainly contribute to the browning. On the basis above, a model elucidating enzymatic browning mechanisms in plant cell cultures was put forward, and effective control approaches were presented.
褐化现象在植物细胞培养中普遍存在，严重阻碍了植物细胞培养的科学研究和广泛应用。到目前为止，由于植物细胞培养中褐化机制尚不清楚，这个问题仍然没有得到很好的控制。本文以两种具有严重褐化现象的典型材料——红豆杉细胞和胀果甘草细胞为研究对象，对其机制进行了研究。我们的结果表明，褐化归因于一种生理酶促反应，糖调控的酚类生物合成在褐化过程中起决定性作用。此外，为了确定参与酶促褐化反应的具体化合物，分析了红豆杉细胞的转录谱和代谢产物，以及从褐变培养基中提取的褐化化合物的紫外扫描和高效液相色谱-质谱（HPLC-MS）图谱，发现苯丙烷途径衍生的黄酮类化合物是主要化合物，并推断杨梅素和槲皮素是褐化反应的主要底物。抑制黄酮类生物合成可以防止褐化的发生，赤霉素（GA3）作为抑制剂通过阻断黄酮类生物合成有效地控制了褐化，这进一步证实了黄酮类化合物是褐化的主要原因。在此基础上，提出了一个阐明植物细胞培养中酶促褐化机制的模型，并给出了有效的控制方法。
