An Ancestral Gibberellin in a Moss Physcomitrella patens
An Ancestral Gibberellin in a Moss Physcomitrella patens
复制标题
DOI：
10.1016/j.molp.2018.03.010
复制
发表时间：
2018-08-06
期刊：
影响因子：
27.5
通讯作者：
Nakajima, Masatoshi
中科院分区：
文献类型：
--
作者：
Miyazaki, Sho;Hara, Mariho;Nakajima, Masatoshi
在开花植物中发现的大多数植物激素有助于苔藓小立碗藓的生长调节。这种苔藓不产生赤霉素（GA）（图1A）。已经在展叶牡丹中鉴定了对映贝壳杉烯合酶（PpCPS/KS）和对映贝壳杉烯氧化酶（PpKO），但与开花植物相反，展叶牡丹没有对映贝壳杉烯酸氧化酶（KAO）同源物（Hayashi et al.，2010;宫崎等人，2011，图1A）。然而，已经注释了可能与GA合成途径相关的几种2-酮戊二酸依赖性双加氧酶（2 ODD）基因（Hirano等人，2007）和对映贝壳杉烯缺陷的展叶假单胞菌突变体（Ppcps/ks）显示出有限的绿线体到茎线体的原丝体细胞分化，这是产生配子体的快速生长细胞。应用对映贝壳杉烯酸（KA），而不是GA，恢复表型。这些结果支持了展叶商陆在KA下游缺乏GA生物合成途径，表明KA代谢产物可能代替GA在展叶商陆中发挥生理作用。在这封信中，我们报道了展叶商陆中由KA生物合成的激素类二萜化合物的鉴定、生物合成和表征。为了检测KA代谢物，我们使用Ppcps/ks突变体建立了一种互补生物测定系统，可在0.0001-1 mM范围内定量KA（补充图1A和1B）。应用KA突变体后，代谢产物提取，分馏通过反相高效液相色谱法（HPLC），并进行测定系统。使用1 mM KA将Ppcps/ks细胞分化的回收率设定为100%，并在级分15和18中检测到细胞分化活性（图1 B和补充图1C）。使用液相色谱电喷雾电离-串联质谱法（LC-ESIMS/MS）测定这些级分中可能的KA代谢物，如先前报道的（宫崎et al.，2015年）的报告。考虑到KA（分子量302）和GA-分解代谢产物GA 34（分子量348），从m/z 299> 299至350> 350监测到带负电荷的离子（图1C），这表明级分18含有m/z 301> 301 [MH]-离子的未代谢KA（图1C）。另一方面，在级分15中发现了可能的候选物：m/z 317> 317 [M + H]-离子，tR 7.8 min（图1C）。假设大于KA的16个m/z离子与氧一起组成KA骨架，而没有任何去饱和。
Most of the plant hormones found in flowering plants contribute to growth regulation in the moss Physcomitrella patens. This moss does not produce gibberellin (GA)(Figure 1 A). An ent-kaurene synthase (PpCPS/KS) and an ent-kaurene oxidase (PpKO) have been identified in P. patens, but in contrast to flowering plants, P. patens has no ent-kaurenoic acid oxidase (KAO) homologs (Hayashi et al., 2010; Miyazaki et al., 2011, Figure 1 A). Nevertheless, several 2-oxoglutarate-dependent dioxygenase (2ODD) genes, which might be related to the GA synthesis pathway, have been annotated (Hirano et al., 2007), and ent-kaurene-deficient P. patens mutants (Ppcps/ks) showed limited protonemal cell differentiation of chloronemata to caulonemata, which are fast-growing cells that produce gametophores. The application of ent-kaurenoic acid (KA), but not GA, recovered the phenotype. These results support that P. patens lacks a GA biosynthetic pathway downstream of KA, indicating that KA metabolite (s) instead of GA may play physiological roles in P. patens.In this letter, we report the identification, biosynthesis, and characterization of a hormonal diterpenoid compound biosynthesized from KA in P. patens. For detecting KA metabolites, we established a complementation bioassay system using Ppcps/ks mutants that quantifies KA within the 0.0001–1 mM range (Supplemental Figure 1A and 1B). After applying KA to the mutant, metabolites were extracted, fractionated by reversedphase high-performance liquid chromatography (HPLC), and subjected to the assay system. The recovery ratio of Ppcps/ks cell differentiation was set to 100% using 1 mM KA, and cell differentiation activities were detected in fractions 15 and 18 (Figure 1 B and Supplemental Figure 1C). Possible KA metabolites in these fractions were determined using liquid chromatographyelectrospray ionization-tandem mass spectrometry (LC-ESIMS/MS), as previously reported (Miyazaki et al., 2015). Considering KA (molecular weight 302) and the GA-catabolite GA34 (molecular weight 348), negatively charged ions were monitored from m/z 299> 299 to 350> 350 (Figure 1 C), which revealed that fraction 18 contained unmetabolized KA with an m/z 301> 301 [M À H]–ion (Figure 1 C). On the other hand, a possible candidate was found in fraction 15: an m/z 317> 317 [M À H]–ion at tR 7.8 min (Figure 1 C). The 16 m/z ions larger than that from KA were assumed to compose the KA skeleton with an oxygen without any desaturation.