Plant Developmental Genetics, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.
Bioscience, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.
Plant J. 2023 Jan;113(1):7-22. doi: 10.1111/tpj.16024. Epub 2022 Nov 28.
Somatic embryogenesis (SE), or embryo development from in vitro cultured vegetative explants, can be induced in Arabidopsis by the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) or by overexpression of specific transcription factors, such as AT-HOOK MOTIF NUCLEAR LOCALIZED 15 (AHL15). Here, we explored the role of endogenous auxin [indole-3-acetic acid (IAA)] during 2,4-D and AHL15-induced SE. Using the pWOX2:NLS-YFP reporter, we identified three distinct developmental stages for 2,4-D and AHL15-induced SE in Arabidopsis, with these being (i) acquisition of embryo identity; (ii) formation of pro-embryos; and (iii) somatic embryo patterning and development. The acquisition of embryo identity coincided with enhanced expression of the indole-3-pyruvic acid auxin biosynthesis YUCCA genes, resulting in an enhanced pDR5:GFP-reported auxin response in the embryo-forming tissues. Chemical inhibition of the indole-3-pyruvic acid pathway did not affect the acquisition of embryo identity, but significantly reduced or completely inhibited the formation of pro-embryos. Co-application of IAA with auxin biosynthesis inhibitors in the AHL15-induced SE system rescued differentiated somatic embryo formation, confirming that increased IAA levels are important during the last two stages of SE. Our analyses also showed that polar auxin transport, with AUXIN/LIKE-AUX influx and PIN-FORMED1 efflux carriers as important drivers, is required for the transition of embryonic cells to proembryos and, later, for correct cell fate specification and differentiation. Taken together, our results indicate that endogenous IAA biosynthesis and its polar transport are not required for the acquisition of embryo identity, but rather to maintain embryonic cell identity and for the formation of multicellular proembryos and their development into histodifferentiated embryos.
体细胞胚胎发生（SE），或体外培养营养体外植体的胚胎发育，可以通过合成生长素 2,4-二氯苯氧乙酸（2,4-D）或通过特定转录因子的过表达来诱导拟南芥，如 AT-HOOK MOTIF NUCLEAR LOCALIZED 15（AHL15）。在这里，我们探讨了内源性生长素[吲哚-3-乙酸（IAA）]在 2,4-D 和 AHL15 诱导的 SE 过程中的作用。使用 pWOX2：NLS-YFP 报告基因，我们鉴定了拟南芥中 2,4-D 和 AHL15 诱导的 SE 的三个不同发育阶段，这些阶段为：（i）获得胚胎身份；（ii）形成原胚；（iii）体细胞胚形态发生和发育。胚胎身份的获得与吲哚-3-丙酮酸生长素生物合成 YUCCA 基因的增强表达同时发生，导致胚胎形成组织中增强的 pDR5：GFP 报告生长素反应。吲哚-3-丙酮酸途径的化学抑制不影响胚胎身份的获得，但显著减少或完全抑制原胚的形成。在 AHL15 诱导的 SE 系统中，将 IAA 与生长素生物合成抑制剂共同应用可挽救分化的体细胞胚形成，这证实了在 SE 的后两个阶段中，IAA 水平的增加是重要的。我们的分析还表明，极性生长素运输，以 AUXIN/LIKE-AUX 内流和 PIN-FORMED1 外流载体为重要驱动因素，对于胚胎细胞向原胚的转变以及随后正确的细胞命运特化和分化是必需的。总之，我们的结果表明，内源性 IAA 生物合成及其极性运输对于胚胎身份的获得不是必需的，而是对于维持胚胎细胞身份以及形成多细胞原胚及其发育成组织分化的胚胎是必需的。
