By Fu-xiang Wang, Guan-dong Shang | September 28, 2020
Plant somatic embryogenesis refers to a phenomenon where embryos develop from somatic cells in the absence of fertilization. Previous studies have revealed that the phytohormone auxin plays a crucial role in somatic embryogenesis by inducing a cell totipotent state, although its underlying mechanism is poorly understood. Here, we show that auxin rapidly rewires the cell totipotency network by altering chromatin accessibility. The analysis of chromatin accessibility dynamics further reveals a hierarchical gene regulatory network underlying somatic embryogenesis. Particularly, we find that the embryonic nature of explants is a prerequisite for somatic cell reprogramming. Upon cell reprogramming, the B3-type totipotent transcription factor LEC2 promotes somatic embryo formation by direct activation of the early embryonic patterning genes WOX2 and WOX3. Our results thus shed light on the molecular mechanism by which auxin promotes the acquisition of plant cell totipotency and establish a direct link between cell totipotent genes and the embryonic development pathway.
Owing to their sessile nature, plants maintain cell pluripotency or totipotency throughout their life cycles. Somatic cells are able to regenerate themselves in response to chemical or mechanical stimuli or go through somatic embryogenesis (SE) to regenerate whole plants (Birnbaum and Sánchez Alvarado, 2008; Ikeuchi et al., 2019, 2016; Sena and Birnbaum, 2010; Sugimoto et al., 2011). SE refers to the development of ectopic embryos from somatic cells independent of gamete formation and fertilization. Since its first documentation in 1950s (Reinert, 1958; Steward et al., 1958; Waris, 1957), SE has become a powerful tool in plant biotechnology for the propagation of endangered species and generation of genetically modified plants with improved traits (Lowe et al., 2016). Thus, the elucidation of the molecular and cellular basis of SE is of great importance to understand the basic principles underlying embryonic patterning and epigenetic reprogramming in plants (Birnbaum and Roudier, 2017; Méndez-Hernández et al., 2019; Palovaara et al., 2016; Radoeva et al., 2019b; Radoeva and Weijers, 2014; Smertenko and Bozhkov, 2014; Winkelmann, 2016; Wójcik et al., 2020; Xu and Huang, 2014).
Somatic embryos can be induced by exposing immature zygotic embryos or male gametophytes to the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) or to abiotic stress (Custers et al., 1994; Fehér, 2015; Gaj, 2011). It has been shown that local auxin biosynthesis and polar auxin transport are essential for the establishment of auxin gradients during somatic embryo formation (Bai et al., 2013; Soriano et al., 2014; Su and Zhang, 2009). How auxin induces cell totipotency and how auxin promotes embryogenesis remains unclear.
Overexpression of certain key transcription factors (TFs) has been used to induce the differentiation of stem cells or SE in both animals and plants. In 2006, Takahashi and Yamanaka showed that a combination of four specific TFs were involved in the conversion of differentiated fibroblasts to a pluripotent state resembling embryonic stem cells derived from the blastocyst inner cell mass (Takahashi and Yamanaka, 2006). Similarly, SE in Arabidopsis can be achieved by ectopic overexpression of a single TF, including the homeodomain TF WUSCHEL (WUS), AP2-domain TF PLETHORA4/BABY BOOM (PLT4/BBM) or PLT5/EMBRYO MAKER (PLT5/EMK), MADS-box TF AGAMOUS-LIKE15 (AGL15), NF-Y (nuclear factor of the Y box) TF LEAFY COTYLEDON1 (LEC1), B3 TF LEC2 and FUSCA3 (FUS3), MYB TF MYB118 and RWP-RK DOMAIN-CONTAINING4 (RKD4)/GROUNDED (GRD) (Boutilier et al., 2002; Gallois et al., 2004; Harding et al., 2003; Lotan et al., 1998; Stone et al., 2001; Thakare et al., 2008; Tsuwamoto et al., 2010; Waki et al., 2011; Wang et al., 2009b; Zuo et al., 2002). In addition, a large number of other TFs that are differentially expressed during SE have been identified through comprehensive time-course analyses (Gliwicka et al., 2013; Szczygieł-Sommer and Gaj, 2019; Wickramasuriya and Dunwell, 2015). The downstream events of these TFs were also analyzed. Transcriptional crosstalk among these different TFs was revealed by genome-wide target identification, and notably, BBM, LEC2, AGL15, and PLT5 were found to regulate common pathways, in particular that of auxin (Braybrook et al., 2006; Horstman et al., 2017b; Pinon et al., 2013; Stone et al., 2008; Zheng et al., 2009). However, how the overexpression of individual TFs from disparate families is able to induce SE is poorly understood, and whether these TFs promote embryogenesis through a common developmental pathway is not known.
As in animals, epigenetic reprogramming also plays an important role in the acquisition of totipotency and SE (Wójcikowska et al., 2020). POLYCOMB REPRESSIVE COMPLEX2 (PRC2), a chromatin regulator that maintains gene repression through the deposition of the histone H3 lysine 27 trimethylation (H3K27me3) marker, constitutes a major barrier to the hormone-mediated establishment of embryogenic competence in mature somatic cells in Arabidopsis (Ikeuchi et al., 2015; Mozgová et al., 2017). Accordingly, the mutation of PRC2 subunits leads to the formation of callus on the shoot apex or in some cases to disorganized cell masses and somatic embryos that develop from single root hairs (Chanvivattana et al., 2004; Ikeuchi et al., 2015). It has been proposed that chemical perturbation or genetic disruption of PRC2 may induce SE through de-repression of the TF genes, such as AGL15, BBM, LEC1, LEC2, and PLT5 (Bouyer et al., 2011; Ikeuchi et al., 2015; Liu et al., 2016; Mozgová et al., 2017). Furthermore, treatment of Arabidopsis explants with trichostatin A (TSA), a chemical inhibitor of histone deacetylases, induces SE without the exogenous application of auxin (Wójcikowska et al., 2018).
To sum up, much progress has been made in the elucidation of the underlying mechanisms of SE. However, our understanding of how auxin, TFs, and epigenetic regulation collaboratively regulate somatic cell fate transition is still limited. Here, we report the chromatin accessibility landscape at the early stage of SE. We find that auxin rapidly induces the acquisition of cell totipotency by altering chromatin accessibility. The analysis of chromatin accessibility and transcriptome dynamics further reveal a hierarchical TF cascade underlying auxin-induced SE. In particular, our results uncover a long sought-after molecular link between cell totipotency genes and the early embryonic development pathway.