Wang Peng, Guo Longyun, Jaini Rohit, Klempien Antje, McCoy Rachel M, Morgan John A, Dudareva Natalia, Chapple Clint
1Department of Biochemistry, Purdue University, West Lafayette, IN 47907 USA.
2Davidson School of Chemical Engineering, Purdue University, West Lafayette, IN 47907 USA.
Plant Methods. 2018 Jun 23;14:51. doi: 10.1186/s13007-018-0318-3. eCollection 2018.
Metabolic fluxes represent the functional phenotypes of biochemical pathways and are essential to reveal the distribution of precursors among metabolic networks. Although analysis of metabolic fluxes, facilitated by stable isotope labeling and mass spectrometry detection, has been applied in the studies of plant metabolism, we lack experimental measurements for carbon flux towards lignin, one of the most abundant polymers in nature.
We developed a feeding strategy of excised Arabidopsis stems with C labeled phenylalanine (Phe) for the analysis of lignin biosynthetic flux. We optimized the feeding methods and found the stems continued to grow and lignify. Consistent with lignification profiles along the stems, higher levels of phenylpropanoids and activities of lignin biosynthetic enzymes were detected in the base of the stem. In the feeding experiments, C labeled Phe was quickly accumulated and used for the synthesis of phenylpropanoid intermediates and lignin. The intermediates displayed two different patterns of labeling kinetics during the feeding period. Analysis of lignin showed rapid incorporation of label into all three subunits in the polymers.
Our feeding results demonstrate the effectiveness of the stem feeding system and suggest a potential application for the investigations of other aspects in plant metabolism. The supply of exogenous Phe leading to a higher lignin deposition rate indicates the availability of Phe is a determining factor for lignification rates.
代谢通量代表生化途径的功能表型，对于揭示前体在代谢网络中的分布至关重要。尽管借助稳定同位素标记和质谱检测对代谢通量进行的分析已应用于植物代谢研究，但我们缺乏针对木质素（自然界中最丰富的聚合物之一）碳通量的实验测量。
我们开发了一种用碳标记的苯丙氨酸（Phe）饲喂拟南芥离体茎的策略，用于分析木质素生物合成通量。我们优化了饲喂方法，发现茎继续生长并木质化。与沿茎的木质化情况一致，在茎基部检测到更高水平的苯丙烷类化合物和木质素生物合成酶的活性。在饲喂实验中，碳标记的Phe迅速积累并用于合成苯丙烷类中间体和木质素。在饲喂期间，中间体表现出两种不同的标记动力学模式。对木质素的分析表明，标记迅速掺入聚合物的所有三个亚基中。
我们的饲喂结果证明了茎饲喂系统的有效性，并表明其在植物代谢其他方面研究中的潜在应用。外源Phe的供应导致更高的木质素沉积速率，这表明Phe的可用性是木质化速率的决定因素。
