Godee Cara, Mira Mohamed M, Wally Owen, Hill Robert D, Stasolla Claudio
Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada.
Permanent address: Department of Botany, Faculty of Science, Tanta University, Tanta, Egypt 31527.
J Exp Bot. 2017 Feb 1;68(5):1013-1023. doi: 10.1093/jxb/erx003.
Mutation of phytoglobin 2 (Pgb2) increases the number of somatic embryos in Arabidopsis. To assess the effects of the cellular localization of Pgb2 on embryo formation, an inducible system expressing a fusion protein consisting of Pgb2 linked to the steroid-binding domain of the rat glucocorticoid receptor (GR) was introduced in a pgb2 mutant line lacking the ability to express Pgb2. In this transgenic system, Pgb2 remains in the cytoplasm but migrates into the nucleus upon exposure to dexamethasone (DEX). Pgb2 retention in the cytoplasm, in the absence of DEX, increased the number of somatic embryos and reduced the expression of MYC2 - an inhibitor of the synthesis of auxin, which is the inductive signal for embryogenesis. Removal of DEX also induced the expression of several genes involved in the biosynthesis of tryptophan and the auxin, indole-3-acetic acid (IAA). These genes included: tryptophan synthase-α subunit (TSA1) and tryptophan synthase-β subunit (TSB1), which are involved in the synthesis of tryptophan, cytochrome P450 CYP79B2 (CYP79B2) and amidase 1 (AMI1), which participate in the formation of IAA via indole-3-acetaldoxime, and several members of the YUCCA family, including YUC1 and 4, which are also required for IAA synthesis. Retention of Pgb2 in the cytoplasm by removal of DEX increased the staining pattern of IAA along the cotyledons of the explants generating embryogenic tissue. Staining for IAA decreased when Pgb2 translocated into the nucleus in response to the application of DEX. Collectively, these results suggest that the presence of Pgb2 in the cytoplasm, but not in the nucleus, phenocopies the effects of Pgb2 mutation in inducing somatic embryogenesis.
植物血红蛋白2（Pgb2）的突变增加了拟南芥体细胞胚的数量。为了评估Pgb2的细胞定位对胚胎形成的影响，在一个缺乏表达Pgb2能力的pgb2突变株系中引入了一个可诱导系统，该系统表达一种融合蛋白，该融合蛋白由与大鼠糖皮质激素受体（GR）的类固醇结合结构域相连的Pgb2组成。在这个转基因系统中，Pgb2保留在细胞质中，但在暴露于地塞米松（DEX）后迁移到细胞核中。在没有DEX的情况下，Pgb2保留在细胞质中增加了体细胞胚的数量，并降低了MYC2的表达，MYC2是生长素合成的抑制剂，而生长素是胚胎发生的诱导信号。去除DEX也诱导了几个参与色氨酸和生长素吲哚-3-乙酸（IAA）生物合成的基因的表达。这些基因包括：参与色氨酸合成的色氨酸合酶-α亚基（TSA1）和色氨酸合酶-β亚基（TSB1），通过吲哚-3-乙醛肟参与IAA形成的细胞色素P450 CYP79B2（CYP79B2）和酰胺酶1（AMI1），以及YUCCA家族的几个成员，包括IAA合成也需要的YUC1和YUC4。通过去除DEX使Pgb2保留在细胞质中增加了IAA在外植体子叶上沿产生胚性组织部位的染色模式。当Pgb2响应DEX的应用而转移到细胞核中时，IAA染色减少。总体而言，这些结果表明，Pgb2存在于细胞质而非细胞核中，模拟了Pgb2突变在诱导体细胞胚胎发生中的作用。
