Department of Life Science, Chung-Ang University, Seoul, 156-756, Republic of Korea.
Physiol Plant. 2015 Jan;153(1):58-67. doi: 10.1111/ppl.12242. Epub 2014 Jul 29.
An in vitro enzyme assay using radioisotope-labeled (3) H-castasterone ((3) H-CS) or (32) P-ATP showed that CS can be phosphorylated by ATP in Arabidopsis and tomato plants. Gas chromatography-mass spectrometry (GC-MS) analysis using non-isotope-labeled CS and ATP revealed that the phosphorylation of CS occurs at the side chain, most likely at the C-23 hydroxyl. The polar fractions than free brassinosteroids (BRs) obtained from extracts of Arabidopsis and tomato showed almost no BRs activity in a rice lamina inclination bioassay. However, the fractions showed increased bioactivity after treatment with wheat germ acidic phosphatase (WGAP). Additionally, CS was identified from the hydrolysate by WGAP using GC-MS analysis in both plants. In contrast, the polar fractions obtained from BR-deficient mutants, Arabidopsis cyp85a2 and tomato d(x) , did not show an increase in biological activity with WGAP treatment, and no free BRs, including CS, were detected in the hydrolysate. This suggests that CS phosphate is a naturally occurring biologically inactive conjugate that is generated when CS is normally synthesized in Arabidopsis and tomato plants. Taken together, these results suggest that phosphorylation of CS is an important conjugation process for the maintenance of the homeostatic level of an active BR and thus the regulation of the growth and development of plants.
利用放射性同位素标记的（3）H-脱落酸（（3）H-CS）或（32）P-ATP 的体外酶分析表明，CS 可以在拟南芥和番茄植物中被 ATP 磷酸化。使用非同位素标记的 CS 和 ATP 的气相色谱-质谱（GC-MS）分析表明，CS 的磷酸化发生在侧链上，很可能在 C-23 羟基上。从拟南芥和番茄提取物中获得的极性分数比游离油菜素内酯（BRs）在水稻叶片倾斜生物测定中几乎没有 BRs 活性。然而，在用小麦胚芽酸性磷酸酶（WGAP）处理后，这些分数显示出生物活性增加。此外，通过在两种植物中使用 GC-MS 分析，在 WGAP 处理的水解物中鉴定出 CS。相比之下，从 BR 缺陷突变体，拟南芥 cyp85a2 和番茄 d（x）中获得的极性分数在用 WGAP 处理后没有显示出生物活性增加，并且在水解物中未检测到游离 BRs，包括 CS。这表明 CS 磷酸盐是一种天然存在的生物活性无活性缀合物，当 CS 在拟南芥和番茄植物中正常合成时会产生。综上所述，这些结果表明 CS 的磷酸化是维持活性 BR 体内平衡水平和调节植物生长发育的重要缀合过程。
