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. 2020 Sep 3;1(3):185–193. doi: 10.1007/s42994-020-00029-8

Regulation of cell reprogramming by auxin during somatic embryogenesis

Li Ping Tang 1, Xian Sheng Zhang 1, Ying Hua Su 1,✉
PMCID: PMC9590521  PMID: 36303566

Abstract

How somatic cells develop into a whole plant is a central question in plant developmental biology. This powerful ability of plant cells is recognized as their totipotency. Somatic embryogenesis is an excellent example and a good research system for studying plant cell totipotency. However, very little is known about the molecular basis of cell reprogramming from somatic cells to totipotent cells in this process. During somatic embryogenesis from immature zygotic embryos in Arabidopsis, exogenous auxin treatment is required for embryonic callus formation, but removal of exogenous auxin inducing endogenous auxin biosynthesis is essential for somatic embryo (SE) induction. Ectopic expression of specific transcription factor genes, such as “LAFL” and BABY BOOM (BBM), can induce SEs without exogenous growth regulators. Somatic embryogenesis can also be triggered by stress, as well as by disruption of chromatin remodeling, including PRC2-mediated histone methylation, histone deacetylation, and PKL-related chromatin remodeling. It is evident that embryonic identity genes are required and endogenous auxin plays a central role for cell reprogramming during the induction of SEs. Thus, we focus on reviewing the regulation of cell reprogramming for somatic embryogenesis by auxin.

Keywords: Somatic embryogenesis, Auxin, Cell reprogramming, Transcription factors, Chromatin remodeling

Introduction

Single-celled zygotes of both plants and animals have the capacity to form a whole organism and are considered totipotent. However, only plants have the capacity to regenerate whole plants from differentiated somatic cells of a variety of tissues through in vitro culture, displaying remarkable developmental plasticity. Gottleib Haberlandt (1854–1945) proposed the concept of plant totipotency in 1902, based on the cell theory of Schleiden (1838) and Schwann (1839). At that time, Haberlandt hypothesized that entire plants could be generated by culturing isolated somatic cells. However, there was not any experimental evidence to support the hypothesis for more than half a century. Then in 1958, Steward showed that entire plants could be regenerated from segments of the differentiated secondary phloem of a carrot (Steward et al. 1958), thus demonstrating the remarkable totipotency of plant cells.

Somatic embryogenesis is an important model system for plant regeneration and is believed to be the strongest proof for the totipotency of plant somatic cells. Somatic embryogenesis refers to the process of asexual reproduction of somatic cells leading to the development of a new individual from vegetative tissues, such as leaf protoplasts (O'Neill and Mathias 1993; Luo and Koop 1997), zygotic embryos (Kobayashi et al. 2010), shoot apices, or flower buds (Ikeda-Iwai et al. 2003). Appropriate inducers, including hormones, molecular regulators and stresses, are necessary for somatic embryogenesis. In recent years, various somatic embryogenesis systems have been established (Williams and Maheswaran 1986; Sangwan et al. 1992; Wu et al. 1992; O'Neill and Mathias 1993; Ikeda-Iwai et al. 2003; Forster et al. 2007; Kobayashi et al. 2010; Seguí-Simarro 2010). Among them, the addition of exogenous auxin is the most common method for inducing somatic embryogenesis from immature zygotic embryos (Sangwan et al. 1992; Wu et al. 1992; Ikeda-Iwai et al. 2002).

Somatic embryogenesis is a complex process. The somatic cells in explants may undergo proliferation, dedifferentiation, and redifferentiation (Elhiti et al. 2013). At the molecular level, a large number of regulators are involved in the cellular reprogramming that occurs during the transition of somatic cells into the embryogenic state to achieve totipotency (Zeng et al. 2007). During this process, it appears that the small molecule auxin plays a central role under appropriate conditions. Thus, we focus on reviewing the regulation of auxin-induced cell reprogramming for somatic embryogenesis.

The role of exogenous hormones

Auxin has been recognized as a potent initiator of somatic embryogenesis in plants. For example, high concentrations of indole-3-acetic acid (IAA), 2-naphthoxyacetic acid (2-NOA), 3-indolebutyric acid (IBA), and 1-naphthylacetic acid (NAA) can induce the formation of somatic embryos (SEs) (Kamada and Harada 1979; Von Arnold and Hakman 1988; Becwar et al. 1989). The most commonly used hormone is the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-d), which displays the highest efficiency of SE induction in a variety of in vitro culture systems (Michalczuk et al. 1992; Charriere et al. 1999; Pasternak et al. 2002; Ikeda-Iwai et al. 2003). Sangwan and Wu reported an Arabidopsis somatic embryogenesis system in which SEs can be induced from immature zygotic embryos by 2,4-d (Sangwan et al. 1992; Wu et al. 1992). SEs can also be generated from intermediate callus of Arabidopsis using 2,4-d (Pillon et al. 1996). In this procedure, immature zygotic embryos are precultured with 2,4-d for embryogenic callus induction, and then SEs are produced from the embryogenic callus following transfer to auxin-free medium (Pillon et al. 1996; Ikeda-Iwai et al. 2002). Regeneration of SEs is severely suppressed by the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) and by inactivating the PIN-FORMED 1 (PIN1) auxin efflux carrier (Su et al. 2009). Disruption of the auxin biosynthetic genes YUCCA 2 (YUC2) and YUC4 results in the production of fewer SEs than wild type in 2,4-d-induced somatic embryogenesis (Wójcikowska et al. 2013; Bai et al. 2013). Moreover, mutants defective in auxin response, such as the auxin-resistant (axr) mutants, display a repressed capacity for somatic embryogenesis (Gaj et al. 2006). Thus, endogenous auxin biosynthesis, polar auxin transport, and auxin responses are required for the induction of SEs by exogeneous auxin.

Besides auxin, several other hormones such as cytokinin, ethylene, abscisic acid (ABA), and gibberellic acid (GA), also function in somatic embryogenesis. In 2,4-d-induced somatic embryogenesis of petunia, SE cannot be formed from embryogenic callus treated with Lovastatin, which is a cytokinin biosynthesis inhibitor (Antofie and Mitoi 2019). Moreover, fewer and rootless SEs are formed when the cytokinin response is suppressed by overexpression of type-A Arabidopsis response regulator (ARR) genes (Su et al. 2015), indicating that cytokinin acts as an important factor for auxin-induced SE formation. In some plant species, such as Norway spruce (Picea abies), exogenous auxin and cytokinin are required for embryogenic callus formation, from which SEs are induced after removal of both hormones (Larsson et al. 2008). Therefore, cytokinin also plays a positive role in the acquisition of embryonic ability by somatic cells. It was reported that the early expression of the WUSCHEL (WUS) and WUSCHEL-RELATED HOMEOBOX5 (WOX5) genes, which are essential for shoot and root meristem initiation, was induced in embryonic callus derived from the auxin-induced immature zygotic embryos (Su et al. 2015). Cytokinin and auxin response patterns play an important role in WUS and WOX5 regional expression and the subsequent embryonic shoot–root axis establishment during somatic embryogenesis (Su et al. 2015).

The hormone ethylene was found to interfere with the formation of SEs (Su and Zhang 2014). Ethylene synthesis and signaling are dramatically downregulated after the removal of 2,4-d for SE (Bai et al. 2013). The induction of SEs is also disrupted by increasing ethylene production in the embryonic callus (Guzmán and Ecker 1990; Wang et al. 2004; Bai et al. 2013). Increased endogenous ethylene levels were reported to suppress the expression of YUC genes and to decrease the level of endogenous IAA (Bai et al. 2013). However, in some cases, ethylene plays a positive role in somatic embryogenesis. For example, an ethylene response gene, encoding the AP2/ERF transcription factor SOMATIC EMBRYO RELATED FACTOR 1 (SERF1), is required for auxin and cytokinin-induced somatic embryogenesis from leaf explants of Medicago truncatula (Mantiri et al. 2008). The evidence provided here suggests that the effects depend on the species of explants. ABA is believed to be positively associated with somatic embryogenesis (Karami et al. 2009; Karami and Saidi 2010; Bai et al. 2013). SEs can be formed from shoot apical tips of carrot with exogenous ABA treatment (Nishiwaki et al. 2000; Kikuchi et al. 2006). A mutation of ABSCISIC ACID DEFICIENT 2 (ABA2), which is an ABA biosynthetic gene, impairs Arabidopsis SE formation (Su et al. 2013). Fluridone, a potent inhibitor of ABA biosynthesis, disrupts the auxin response pattern in embryogenic callus and inhibits SE initiation. GA, on the other hand, has negative effects on somatic embryogenesis. GA biosynthesis inhibitors enhance somatic embryogenesis in conifers (Pullman et al. 2005). The transcript level of GIBBERELLIN 20-OXIDASE (GA20OX), which is involved in GA inactivation, is significantly upregulated during the induction of SEs (Elhiti et al. 2010).

Functions of embryonic identity transcription factors

Ectopic overexpression of some embryonic identity transcription factor genes in Arabidopsis can induce the formation of SEs without exogenous growth regulators. The transcription factors belong three subgroups: (1) HAP3 family of CCAAT-binding factors LEAFY COTYLEDON 1 (LEC1) and LEC1-LIKE (L1L); (2) a subgroup of the plant-specific B3 domain proteins including ABSCISIC ACID INSENSITIVE 3; and (3) LEAFY COTYLEDON 2 (LEC2). LEC1/L1L, ABI3, FUS3, and LEC2 are all named as LAFL (Jia et al. 2013). The LAFL genes are mainly expressed as major regulators throughout embryogenesis, and are required for embryonic identity (Giraudat et al. 1992; Lotan et al. 1998; Luerßen et al. 1998; Stone et al. 2001; Kwong et al. 2003).

Ectopic expression of LEC1, LEC2, and L1L /NUCLEAR FACTOR Y subunit B6 (NF-YB6) in vegetative somatic cells gives rise to SEs from Arabidopsis seedlings (Lotan et al. 1998; Stone et al. 2001; Mu et al. 2013). The loss-of-function mutations in LEC1 and LEC2 strongly impair the auxin-induced formation of SEs (Gaj et al. 2005). Overexpression of LEC1 triggers SE formation, which requires endogenous auxin accumulation (Casson and Lindsey 2006). LEC1 induces auxin biosynthesis via activating the expression of YUC10 (Junker et al. 2012). LEC2 activates the expression of YUC2 and YUC4, as well as TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1), another auxin biosynthetic gene (Stone et al. 2008; Zhao 2014). As a result, overexpressing LEC1 or LEC2 increases the levels of endogenous IAA, thereby reducing the requirement of exogenous 2,4-d in embryonic callus induction (Gaj et al. 2005; Wójcikowska et al. 2013). On the other hand, LEC2 directly activates the expression of AGAMOUS-LIKE 15 (AGL15), which encodes a MADS box transcription factor. Overexpression of AGL15 enhances the ability of SE induction from immature zygotic embryos by exogenous auxin (Harding et al. 2003; Braybrook et al. 2006). These results indicate that overexpression of LEC2 promotes the embryonic cell fate acquisition from somatic cells by directly enhancing AGL15 expression. Moreover, LEC2 and AGL15 upregulate the expression of a noncanonical Aux/IAA gene INDOLE-3-ACETIC ACID INDUCIBLE 30 (IAA30) (Braybrook et al. 2006), which is required for 2,4-d-induced SE formation. Mutation of IAA30 compromises this process (Zheng et al. 2009). Overexpression of FUS3 in epidermal cells leads to the formation of cotyledon-like leaves from the shoot meristem, and FUS3 expression can be induced by auxin (Gazzarrini et al. 2004). In lateral root formation, LEC2 and FUS3 interact to activate YUC4 transcription through directly binding to its promoter. Thus, overexpression of both genes may increase endogenous auxin biosynthesis by increasing YUC4 expression (Tang et al. 2017). In lec1 lec2, lec1 fus3 or lec2 fus3 double and in fus3 lec1 lec2 triple mutants, 2,4-d-induced somatic embryogenesis was completely repressed (Gaj et al. 2005, 2006). Therefore, these embryonic identity genes are required for SE formation induced by exogenous auxin, possibly through their activation of endogenous auxin biosynthesis, which is the key factor for initiating cell totipotency in somatic cells.

Another transcription factor, BABY BOOM (BBM) can induce somatic embryogenesis when expressed ectopically in seedlings of various species (Boutilier et al. 2002; Morcillo et al. 2007; Deng et al. 2009; El Ouakfaoui et al. 2010; Heidmann et al. 2011; Lutz et al. 2011; Bandupriya et al. 2014; Yang et al. 2014; Florez et al. 2015; Lowe et al. 2016). A recent study demonstrated that BBM activates the expression of LAFL genes and AGL15 by directly binding to their promoters (Horstman et al. 2017a). Consequently, the loss-of-function mutations of these LAFL genes significantly repress the BBM-mediated somatic embryogenesis. Therefore, BBM converts somatic cells into embryogenic, totipotent cells through transcriptional activation of the LAFL genes. However, a reduced BBM expression is seen in the embryos of lafl mutants, implying that LAFL proteins in turn positively regulate BBM expression (Horstman et al. 2017a). Similar to LAFL genes, BBM can directly bind to the promoters of YUC3, YUC8 and TAA1 to activate their expression and to increase auxin biosynthesis (Horstman et al. 2017a). Therefore, BBM and LAFL proteins function in a positive feedback regulation loop for promoting cell reprogramming (Fig. 1), possibly through activating the expression of the downstream TAA1 and YUC genes.

Fig. 1.

Fig. 1

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The plant hormone auxin plays a key role as the linker between SE inducing factors and cell reprogramming during somatic embryogenesis. SEs can be regenerated from somatic cells in response to various plant hormones, stress treatments, overexpression of transcription factors, or mutations of the specific chromatin remodelers. 2,4-d is thought to be the most effective inducer for somatic embryogenesis. Auxin biosynthesis is activated, and auxin gradients are re-established in 2,4-d-induced SE generation. Overexpression of transcription factors “LAFL” and BBM induces somatic embryogenesis, which triggers auxin biosynthesis. Mutations of the specific chromatin remodeling factors are also effective to trigger somatic embryogenesis through activation of auxin biosynthesis. For induction of SEs, endogenous auxin is of primary importance, promoting the cell fate transition of somatic cells to totipotent cells and the evocation of SEs

The negative regulation of chromatin remodeling factors

Somatic embryogenesis is involved in activation of key embryonic identity genes, causing target gene expression and subsequently leading to the reprogramming of somatic cells. It was reported that the transcription of these embryonic identity genes is epigenetically regulated, allowing them to be activated or repressed in certain cells at the appropriate developmental stage (Ikeuchi et al. 2015). Besides ectopic expression of the above-mentioned specific transcription factors, loss-of-function mutations of several chromatin remodeling factors induce the formation of SEs in Arabidopsis. In animals, the function of the histone methyltransferase complex POLYCOMB REPRESSIVE COMPLEX 2 (PRC2) was first shown to be required for stem cell pluripotency. PRC2 proteins deposit K27me3 marks on histone H3 for the transcriptional repression of target genes. In Arabidopsis, double mutants of the PRC2 subunit genes, including CURLY LEAF (CLF), SWINGER (SWN) or VERNALIZATION 2 (VRN2), and EMBRYONIC FLOWER 2 (EMF2), SE formation was observed in the double mutants (Chanvivattana et al. 2004). It was found that PRC2 can repress the expression of embryonic identity genes, including AGL15, FUS3, ABI3, and AIL6/PLT3 (Bouyer et al. 2011; Liu et al. 2016). The loss-of-function mutation of PRC2 subunit genes results in significant upregulation of these embryonic identity genes and leads to SE induction. Exogenous 2,4-d induces embryonic callus and subsequent SE formation from immature zygotic embryos with low expression of PRC2. However, SEs can hardly be induced from tissues that highly express PRC2, such as shoots or roots, possibly due to the suppression of embryonic identity gene expression in these differentiated tissues (Liu et al. 2016; Mozgová et al. 2017). Nevertheless, in clf swn double mutants, SEs can be more efficiently induced from the shoot apex when treated with 2,4-d or a combination of 2,4-d and wounding (Mozgová et al. 2017).

Histone acetylation is positively associated with embryogenic reprogramming of plant somatic cells (Tanaka et al. 2008), as the genes encoding histone acetyltransferases are upregulated, while histone deacetylase genes are repressed during SE induction in Arabidopsis (Wickramasuriya and Dunwell 2015). In a recent study, it was shown that Trichostatin A (TSA)-mediated inhibition of histone deacetylases induces the formation of SEs from immature zygotic embryos of Arabidopsis in the absence of exogenous auxin (Wójcikowska et al. 2018). An increased embryogenic response, significantly increased accumulation of endogenous auxin, and enhanced auxin responses were observed in the TSA-treated explants. The embryonic identity genes LEC1, LEC2, AGL15, FUS3 and BBM were also upregulated by TSA treatment. As a result, the expression of YUC1 and YUC10, was also increased (Wójcikowska et al. 2018). Loss-of-function mutation of another chromatin remodeling factor PICKLE (PKL) results in the maintenance of embryonic identities after germination and the pkl mutant forms SEs from seedlings, due to the increased expression of the LEC genes (Ogas et al. 1999; Henderson et al. 2004). The results indicate that repression of either PRC2 or histone deacetylation activity is sufficient to highly induce the expression of embryonic identity genes. Consequently, somatic cells are reprogrammed into totipotent cells, through increased endogenous auxin accumulation.

The effect of stress conditions

The stress conditions used to evoke or promote SE formation include osmotic stress, heavy metals, temperature, ultraviolet radiation, chemical treatments, and wounding (Fehér 2015). Stress conditions can affect auxin homeostasis including auxin biosynthesis, transport and stability (Tognetti et al. 2012). The stress hormones ABA and ethylene interfere with endogenous auxin during the formation of SE (Jiménez et al. 2005; Su and Zhang 2014). Wounding can induce callus formation to prevent infection and water loss (Shigo 1982). Wound-induced callus can regenerate into various tissues and organs as well as SEs, indicating their pluripotency and totipotency (Ikeuchi et al. 2013). Cell reprogramming in callus caused by wounding is regulated by the AP2/ERF transcription factors WOUND INDUCED DEDIFFERENTIATION1 (WIND1), WIND2, WIND3, and WIND4 (Iwase et al. 2011a, b; Ikeuchi et al. 2013). The ectopic overexpression of WIND1 induces embryonic callus formation and SE formation, indicating that totipotent cells were formed in wound-induced callus (Iwase et al. 2011b; Ikeuchi et al. 2013). The ectopic overexpression of WIND transcription factors activates cytokinin signaling in callus formation (Iwase et al. 2011b; Ikeuchi et al. 2013), but not auxin biosynthesis and signaling. However, it is interesting that the embryonic callus induced by ectopic WIND1 expression through upregulation of cytokinin signaling that has similar expression pattern to those induced by high levels of auxin (Iwase et al. 2011b). Moreover, overexpression of both WIND1 and LEC2 induces more embryogenic callus than activation of WIND1 or LEC2 alone (Ikeuchi et al. 2013). These results indicate that WIND1 may interact with the LEC pathway, enhancing auxin signaling in the embryonic callus for the formation of totipotent cell.

Conclusions and perspectives

Under appropriately inductive conditions, differentiated somatic cells can be reprogrammed into totipotent cells and initiate SEs that roughly follow zygotic embryogenesis. This is exemplified by ectopic SE formation and subsequent regeneration of entire plants from somatic cells in response to various plant hormones, stress treatments, overexpression of transcription factors or mutations of specific chromatin remodeling genes (Fig. 1). Although much progress has been made, the molecular mechanisms of somatic embryogenesis have still not been fully resolved. For induction of SE formation, endogenous auxin is of primary importance (Fig. 1). However, enhanced endogenous auxin biosynthesis is not sufficient for spontaneous SE formation from seedlings. It seems that the expression of embryonic identity genes and endogenous auxin accumulation are both required for the somatic-to-totipotent reprogramming of cells in somatic embryogenesis. The embryo identity genes determine the accumulation of embryonic competence in somatic cells, which also need to accumulate high levels of auxin to promote the cell fate transition to totipotent cells and the evocation of SEs.

Many questions remain to be addressed on the totipotency acquisition of somatic cells. Numerous studies have shown that even fully differentiated cells can be reverted to a totipotent state. However, SEs arise from embryonic or meristematic tissues more easily. For example, SEs can easily be induced from the cotyledons or shoot meristem of plants with ectopic expression of the “LAFL” transcription factors or BBM (Horstman et al. 2017b), but hardly from differentiated leaves or roots. What are the repressing factors in those differentiated tissues that inhibit the reprogramming of somatic cells? Even in embryonic or meristematic tissues, not all the cells can be induced to obtain totipotency. Which cells have the potential to be converted into totipotent cells, and what regulatory mechanisms enable these cells to generate SEs? Plant cells have the powerful ability to become totipotent when they accumulate high enough levels of auxin. What are the mechanisms underlying auxin’s control of the reprogramming of somatic cells to acquire totipotency? It is likely that auxin plays a key role as the linker between SE inducing factors and the network controlling the totipotency program, or it may regulate the downstream gene expression required for the initiation of totipotency. As auxin-mediated pathways lead to the activation of the embryogenic program, and the upregulation of embryonic genes triggers the increase of auxin biosynthesis, it will be intriguing to further analyze the feedback regulation between auxin signaling and embryonic gene expression.

Acknowledgements

This work was funded by the National Natural Science Foundation of China (31670320, 31700248) and the Natural Science Foundation of Shandong Province (ZR2017JL016).

Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

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