TRPLSC 2304 No.

of Pages 9

Trends in
Plant Science
Opinion

Towards a hierarchical gene regulatory network

underlying somatic embryogenesis
Fu-Xiang Wang, 1 Guan-Dong Shang, 1,2 and Jia-Wei Wang 1,3,
*

Genome-editing technologies have advanced in recent years but designing Highlights

future crops remains limited by current methods of improving somatic embryo- Chromatin status influences cellular
genesis (SE) capacity. In this Opinion, we provide an update on the molecular competence for somatic embryogenesis.
event by which the phytohormone auxin promotes the acquisition of plant cell to-
Auxin induces the acquisition of totipo-
tipotency through evoking massive changes in transcriptome and chromatin ac- tency through altering chromatin acces-
cessibility. We propose that the chromatin states and individual totipotency- sibility and the transcriptome.
related transcription factors (TFs) from disparate gene families organize into a hi-
The LEC2–WOX2/3 axis serves as a mo-
erarchical gene regulatory network underlying SE. We conclude with a discus-
lecular link between totipotency-related
sion of the practical paths to probe the cellular origin of the somatic embryo transcription factor genes and early em-
and the epigenetic landscape of the totipotent cell state in the era of single-cell bryonic development pathway.
genomics.

Direct and indirect routes for SE

Regeneration is the process of renewal, restoration, and organogenesis. Owing to their sessile
nature, plants have evolved several ways of regeneration, including the natural process of tissue
repair and organ regeneration after wounding and the induced regeneration of organs and
embryos in vitro. Plant non-zygotic embryogenesis (NZE) (see Glossary) refers to the phe-
nomenon where embryos develop from plant cells in the absence of fertilization [1–5]. Based
on the tissue/cell of origin, NZE can be classified into SE (embryos derived from somatic cells),
microspore embryogenesis (embryos derived from haploid microspore cells) [6], apomixis
(embryos derived from haploid or diploid cells in the ovule; e.g., megaspore mother cells,
aposporous initial cells) [7,8], and suspensor embryogenesis (embryos derived from the suspensors)
[9]. It should be noted that the cellular origin of somatic embryos has not yet been solved (see also
‘Future perspectives’) [10]. The ‘somatic’ embryos can be derived not only from the differentiated
somatic cells but also from the meristematic cells of the explants, particularly in the case where
SE is achieved by culturing shoot tips [11,12].
1
National Key Laboratory of Plant
Molecular Genetics (NKLPMG), CAS
Elucidation of the molecular and cellular basis of SE is of great importance to understand the
Center for Excellence in Molecular Plant
basic principles underlying cell reprogramming, differentiation, and morphogenesis in plants. Sciences, Institute of Plant Physiology
Particularly, in combination with genome-editing tools, this knowledge can be used to increase and Ecology (SIPPE), Chinese Academy
of Sciences (CAS), 200032 Shanghai,
plant transformation rates and expand the populations of transformable plants. In this Opinion,
PR China
we mainly focus on recent advances in the understanding of SE in the model plant arabidopsis 2
University of Chinese Academy of
(Arabidopsis thaliana). Sciences (UCAS), Shanghai 200032,
PR China
3
ShanghaiTech University, Shanghai
There are various types of explants that can be used to induce somatic embryos, including 200031, PR China
cotyledons, petioles, leaves, roots, shoot tip, and zygotic embryos [11,13]. At a practical level,
high regenerative capacity for SE is most frequently displayed by zygotic embryos at more
advanced developmental stages in arabidopsis [11]. SE can be achieved by distinct routes;
namely, direct SE, indirect SE, and stress-induced SE (Figure 1) [13]. In the direct SE route, somatic *Correspondence:
embryo formation can be readily induced by culture of immature embryos at the late bent- jwwang@sippe.ac.cn (J.-W. Wang).

Trends in Plant Science, Month 2022, Vol. xx, No. xx https://doi.org/10.1016/j.tplants.2022.06.002 1

© 2022 Elsevier Ltd. All rights reserved.
 Trends in Plant Science

cotyledon stage of development on medium supplemented with the synthetic auxin 2,4- Glossary
dichlorophenoxyacetic acid (2,4-D). Subculture of explants on auxin-free medium induces em- Abscisic acid (ABA): an isoprenoid
bryo development (Figure 1) [14]. plant hormone that functions in many
aspects of plant growth, development,
and stress responses.
In the indirect route, SE is achieved through embryogenic (embryo forming) callus (Figure 1) [13]. Auxin [indole-3-acetic acid (IAA)]:
Although somatic embryos originating from the direct and indirect paths are morphologically one of the phytohormones that play
similar, somaclonal variation often occurs in embryos derived from the indirect SE route and is critical roles in plant development and
regeneration. 2,4-D is a synthetic auxin.
probably due to longer treatment with auxin [15]. Stress-induced SE is the third route for SE
Cell reprogramming: usually refers to
(Figure 1) [16]. In this protocol, somatic embryos can be induced by a pulse treatment of the regression of a differentiated cell to
shoot-apical-tip explants with osmotic stress followed by culture on 2,4-D medium. However, the totipotent or pluripotent state,
as mentioned earlier, these somatic embryos are likely to be derived from the meristematic resulting in cells with stem cell properties.
Chromatin accessibility: the level of
cells of the shoot tip rather than differentiated somatic cells.
physical compaction of chromatin in the
eukaryotic genome. Accessible or open
The totipotency-related TFs and epigenetic barriers for SE in arabidopsis regions are regarded as the genomic
SE in arabidopsis can be achieved by ectopic overexpression of a single TF belonging to dispa- regions that are accessible by nuclear
macromolecules.
rate families. The representative TFs are AP2-domain PLETHORA (PLT) TFs, NF-Y (nuclear factor Explant: part of a plant cultured on
of the Y box) TF LEAFY COTYLEDON1 (LEC1), B3 TF LEC2, RWP-RK DOMAIN-CONTAINING4 growth media.
(RKD4)/GROUNDED (GRD), AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED15 Gene regulatory network: a collection
(AHL15), and WOX TF WUSCHEL (WUS). Given their ability to induce SE, all of these TFs can of genes that interact with each other to
control gene expression levels, which in
be considered totipotency-related TFs [17–22]. Notably, simultaneous overexpression of the turn determine the function of a cell.
orthologs of maize BABY BOOM (BBM/PLT4) and WUS can enhance the transformation rate Non-zygotic embryogenesis (NZE):
in maize, sorghum, sugarcane, and rice [23]. the phenomenon where embryos
develop from plant cells in the absence
of fertilization.
Although the genomic DNA is the same in all of the diploid somatic cells of a plant, not every cell is Single-cell RNA-seq (scRNA-seq):
committed to becoming an embryo. This phenomenon implies that regenerative competence is the technologies that allow the
not determined by DNA sequence per se, but rather by the different epigenetic factors that dissection of gene expression at
single-cell resolution.
might underly cell competence, of which one is the chromatin status of different loci. Moreover,
Somatic embryogenesis (SE): the
physiological cell states, including endogenous hormone content and cell–cell contacts, and phenomenon where embryos are
cell-type-specific expression patterns can also be responsible for restricting cellular competence derived from differentiated somatic cells
for SE. instead of zygotes.
Totipotency: the ability of a single cell
to divide and develop into a complete
As in animals, histone modification plays an important role in the acquisition of totipotency during organism.
SE in plants [24]. Growing lines of evidence reveal that deposition of the repressive histone H3
lysine 27 trimethylation (H3K27me3) marker constitutes one of the epigenetic barriers to the
auxin-induced acquisition of embryogenic competence in differentiated somatic cells [25,26].
Interference with this process leads to the formation of callus on the shoot apex or somatic
embryos on root hairs in arabidopsis. At the molecular level, chemical perturbation or genetic
disruption of H3K27me3 deposition is likely to induce somatic embryo formation through de-
repression of the totipotency-related TF gene loci.

By joint profiling of chromatin accessibility and gene expression, researchers have recently
proposed that the decline of regenerative competence for SE on seed germination is likely to
be caused by the developmentally regulated removal of the permissive chromatin signature at
totipotency-related TF gene loci (Figure 2) [27]. The expression of these TFs can be re-induced
more easily by 2,4-D in immature zygotic embryos than in the post-embryonic explants. This find-
ing thus provides a plausible explanation for why immature zygotic embryos are commonly used
as explants for SE [14]. However, it should be noted that the chromatin accessibilities of
totipotency-related TFs at the seedling stage were accessed only through a bulk assay for
transposase-accessible chromatin sequencing (ATAC-seq). Therefore, it remains possible that
a few somatic cells retain permissive chromatin signatures at the totipotency-related TF gene

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Trends in Plant Science

(A)
2,4-D No 2,4-D

Direct SE

Immature zygotic embryo Proembryogenic masses -D Primary

2,4
somatic embryos
(B)

2,4-D

indirect SE No 2,4-D

Immature zygotic embryo Embryonic callus Somatic embryos

(C)
AHL15/BBM/LEC2/RKD4/WUS

Direct SE

Seedling Somatic embryos

(D)
Osmotic stress

0.7 M Mannitol 2,4-D No 2,4-D

Shoot-tip explant Shoot-tip callus Somatic embryos

Seedling
Trends in Plant Science

Figure 1. Direct and indirect routes for somatic embryogenesis (SE). Four regeneration routes for SE are shown. (A) Direct route [2,4-dichlorophenoxyacetic acid
(2,4-D) induced]: somatic embryo formation is induced by culturing immature arabidopsis embryos at the late bent-cotyledon stage of development on medium
supplemented with 2,4-D. Explants with embryo-like protuberances are further subcultured on auxin-free medium to induce embryo development. (B) Indirect route:
embryogenic (embryo forming) callus is induced by treating explants with 2,4-D. The development of proembryogenic masses on the surface or in the callus mass
precedes the development of single cells or cell clusters into embryos. (C) Direct route [cell-totipotency-related transcription factor (TF) induced]: SE can be achieved
by overexpression of cell-totipotency-related TF genes (e.g., BBM, LEC2, RKD4, WUS, AHL15). (D) Stress-induced SE on the vegetative tissue of arabidopsis. Shoot-
tip explants were first exposed to osmotic stress and then cultured on medium supplemented with 2,4-D. Somatic embryos were induced from the resultant calli on re-
moval of 2,4-D. Abbreviation: SAM, shoot apical meristem.

loci after germination. In this scenario, these regeneratively competent cells may serve as a
pool for the progenitors of SE in the post-embryonic explants (see also ‘Future perspectives’).
Nevertheless, the positive correlation between chromatin accessibility and regenerative capacity
strongly indicates that the accessibility of totipotency-related TF genes for transcription is a major
competence factor for SE.

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 Trends in Plant Science

Explants
Permissive chromatin environment Closed chromatin

BBM/LEC1/LEC2
Totipotency-related TF genes
Early stages high 2,4-D

C h ode
rem
rom ller
ABA
Fs
AR

ati s
n
Stress genes
Chromatin
remodellers

ARFs
BBM/LEC1/LEC2
Totipotency-related TF genes Remodeling genes

Late stages

BBM LEC2
YUCCAs auxin
AGL15 LEC1

Chromatin
remodellers

WOX2/3 Remodeling genes

Embryonic developmental pathway

Trends in Plant Science

Figure 2. An updated hierarchical mechanism for somatic embryogenesis (SE). A permissive chromatin
environment at the loci of cell-totipotency-related transcription factor (TF) genes (e.g., LEC1, BBM, LEC2) is a prerequisite
for somatic cell reprogramming. On 2,4-dichlorophenoxyacetic acid (2,4-D) treatment, these genes are rapidly induced,
probably by auxin response factors (ARFs) (broken line). The cell-totipotency-related TFs can in turn induce YUCs and
auxin biosynthesis, thereby forming a feed-forward loop to reinforce cell-fate transition. LEC2 acts at the output node of
the cell totipotent gene network by direct activation of early embryonic development genes, including WOX2 and WOX3.
In parallel with this, 2,4-D can induce a transcriptional stress response and massive chromatin remodeling of explants (broken
lines). Abscisic acid (ABA) acts downstream of the stress response and may enhance SE through the totipotency-related TFs.
The auxin-directed remodeled genes, together with WOX2 and WOX3, induce somatic embryo formation.

Auxin evokes extensive changes in transcriptome and chromatin accessibility at

the early stage of direct SE
At the molecular level, auxin evokes direct SE at both transcriptional and epigenetic levels
(Figure 2). Increased transcript levels of totipotency-related TFs, including BBM, LEC1, and

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Trends in Plant Science

LEC2, were observed within 4 h after transfer of explants to 2,4-D [27]. It is well known that auxin
induces downstream signaling events by auxin response factor (ARF) TFs [28–31]. The presence
of putative ARF-binding motifs in the promoter regions of BBM and LEC2 suggests that the effect
of ARFs on the totipotency-related TFs genes is direct [27,32]. However, the identities of these
ARFs are currently unknown. Expression analysis revealed that six ARFs (ARF5, ARF6, ARF8,
ARF10, ARF16, and ARF17) were significantly upregulated, whereas five other genes (ARF1,
ARF2, ARF3, ARF11, and ARF18) were substantially downregulated in SE-induced arabidopsis
explants [33]. Evaluation of SE efficiency from the ARF mutants further suggests that seven
ARFs, particularly ARF5, are involved in SE induction. Thus, it is likely that multiple ARFs redun-
dantly contribute to SE in arabidopsis.

In addition to inducing totipotency-related TFs, auxin evokes massive changes in chromatin

accessibility. Time-course ATAC-seq experiments suggest that auxin reprograms chromatin
accessibility in explants rapidly, and the process is largely complete after 3 days [27]. However,
it should be noted that 3 days of 2,4-D exposure is not sufficient to induce SE (C. Philipsen,
PhD thesis, Leiden University, 2017), implying that 2,4-D may also play a role in stabilizing cell-
fate transition at late stages. The acute effect of auxin at the chromatin level (i.e., within a few
hours on exposure to 2,4-D) is likely to be mediated by ARFs, although the precise molecular
mechanism is currently unknown. One possibility, according to a model in the floral meristem pro-
posed by Wu et al. [34], is that an unknown Aux/IAA-ARF complex binds to totipotency-related
TF gene loci in somatic cells. The subsequent recruitment of the TOPLESS-histone deacetylase
complex prevents totipotent gene expression. On auxin treatment, the rapid degradation of
Aux/IAA proteins results in the eviction of TPL-histone deacetylase, recruitment of the chromatin
remodeling complex, and chromatin openness at target genomic DNA near ARF-bound sites. In
favor of this hypothesis, the formation of somatic embryos was significantly affected in tpl and
histone deacetylase mutants [35–37].

Motif-enrichment analysis also indicates that calmodulin-binding transcription activator1

(CAMTA1) and WRKY TF-binding motifs are overrepresented at the very early stage of SE [27].
Because WRKY TFs are key immune regulators in plants [38], while CAMTAs act as master
TFs for salicylic acid biosynthesis [39,40], it is possible that the high-auxin environment rapidly
evokes a general stress response in the explants. A previous study revealed that half of the TFs
that are induced by 2,4-D during the initiation of SE are stress related [41]. In particular, the abiotic
stress hormone abscisic acid (ABA) has been identified as a downstream signaling component at
the intersection between 2,4-D- and stress-induced SE [12,42–44]. In support of this conclusion,
the embryogenic capacity of the explant is enhanced by dehydration and osmotic stresses [16].
Totipotency-related TFs such as LEC1 and LEC2 were initially identified because of their role in
seed maturation and their dependency on ABA [17,18].

An updated hierarchical gene regulatory network underlying direct SE

Based on all of these achievements, we propose that the chromatin states, totipotency-related
TFs, and a large number of other TFs that are differentially expressed during SE [41,45] organize
into a hierarchical mechanism for cell reprogramming of direct SE (Figure 2). (i) Chromatin states
at the top of the hierarchy. The developmental stages contribute to the regenerative competence for
SE by creating the permissive chromatin signature at totipotency-related TF gene loci. (ii) Because
auxin induces the expression of totipotency-related TF genes, including BBM, LEC1, and LEC2,
within a few hours on transfer to medium supplemented with 2,4-D, ARFs constitute second-tier
regulators. Notably, auxin can also trigger massive changes in chromatin accessibility and stress
response at the early stages of SE. (iii) The totipotency-related TFs act as third-tier regulators [46].
Among them, BBM acts upstream of LEC1 and LEC2 since BBM-induced embryogenesis is

Trends in Plant Science, Month 2022, Vol. xx, No. xx 5

 Trends in Plant Science

dependent on transcriptional activation of LEC1 or LEC2, whereas LEC2 functions at output nodes Outstanding questions
to initiate early embryogenesis. In particular, all of these TFs are able to induce the genes involved in What is the cellular origin of somatic
auxin biosynthesis, thereby creating a feedforward loop that reinforces cell-fate transition. (iv) The embryos?
early-embryonic TFs WOX2 and WOX3 serve as the fourth-tier regulators, which initiate somatic
Whether every somatic cell can be
embryo formation directly [27]. WOX2 and WOX3 are direct targets of LEC2; the wox2 wox3 double committed to the totipotent cell state?
mutation nearly abolished somatic embryo formation. Notably, expression and mutant analyses re-
vealed that YUC-dependent IAA biosynthesis is required for the maintenance of embryo identity and What are the precise molecular
features of the totipotent cell state?
growth at this stage [47].
How auxin, TFs, and epigenetic
In the hierarchy, TFs at higher levels differ from those in the lower levels in their ability to induce regulation collaboratively evoke
somatic cell division and fate
SE in the absence of auxin (Figure 2). Overexpression of a single high-level TF gene, such as
transition at the cellular level?
BBM or LEC2, is sufficient to induce SE without auxin [46]. By contrast, the low-level TF
genes WOX2 and WOX3 are necessary but not sufficient to induce somatic embryo formation
[27]. The AT-hook TF AHL15 acts downstream of BBM and is required for efficient BBM-in-
duced SE [22]. Intriguingly, overexpression of AHL15 causes a reduction in heterochromatin
condensation and simultaneously induces polyploidy at the early stage of SE, suggesting
that AHL15 may play an important role in shaping chromatin architecture. It remains unclear
how the MADS-box TFs (AGL15 and AGL18), MYB TF (MYB118), and RKD TF (RKD4) are in-
tegrated into the abovementioned hierarchical network. One possible molecular link is that the
genes are involved in auxin biosynthesis or signaling. In support of this notion, the auxin recep-
tor gene TRANSPORT INHIBITOR RESPONSE1 was identified as a direct target of AGL15 [48].
Another potential linker is the totipotency-related TF itself. Because RKD4 is expressed at a
very early developmental stage of zygotic embryos [19,49], it is possible that RKD4 induces
SE by directly activating the expression of BBM, LEC1, or LEC2. However, the relative exper-
imental evidence is still lacking.

In contrast to the aforementioned gene regulatory network underlying direct SE, WUS plays an
important role in indirect SE in arabidopsis [50,51]. However, the transcriptional dynamics and
epigenetic landscape during indirect SE remain unknown. How WUS and other totipotency-
related TFs are assembled into a regulatory network awaits further investigation.

Concluding remarks and future perspectives

Towards the cellular origin of the somatic embryo
The process of SE starts from a single cell or a group of cells with similar morphology and genetic
background. The identity of the progenitor cells in explants is poorly understood. It has been
shown, by histological techniques, that these totipotent cells differ from the surrounding somatic
cells in five aspects: (i) high nucleus-to-cytoplasm ratio; (ii) a nucleus with a single large nucleolus;
(iii) low heterochromatin level; (iv) the presence of fragmented vacuoles; and (v) symplasmic
isolation [52,53]. However, the marker genes for the progenitor cells in SE have not been
identified yet.

One difficulty in the identification of the progenitor cells at the molecular level is due to the fact
that explants can precede SE from different cellular origins via distinct regeneration routes. For
example, histological studies have found that arabidopsis somatic embryos display a single-cell
or multicellular origin. The cells in both the protodermis and the subprotodermis of the adaxial
side of cotyledons are regeneratively competent [54]. By contrast, somatic embryos can also
be derived from the cells residing in the boundary domain between the shoot apical meristem
and cotyledons [27,55]. Another difficulty is likely to be caused by the nonsynchronous nature
of SE. The differences in the efficiencies of the epigenetic reprogramming of somatic cells from
distinct lineages and the variations in the cell division rate of committed totipotent cells or their

6 Trends in Plant Science, Month 2022, Vol. xx, No. xx

Trends in Plant Science

progenies lead to failure in probing specific genes associated with cell-fate transition, particularly
at the very early stage when only rare progenitor cells of SE exist.

A comparative analysis revealed that SE-forming explants harbor a mixed molecular identity
(shoot apical meristem, root, and embryo) and share a high degree of similarity in their
transcriptome with non-SE-forming explants, except for a limited number of embryonic and
root identity genes that might be involved in the specification of SE [55]. In contrast to bulk
RNA-seq, single-cell RNA-seq (scRNA-seq) enables the characterization of plant cell hetero-
geneity at unprecedented resolution [56–60]. During the past 2 years, scRNA-seq has been
widely used to define the cellular taxonomy of the arabidopsis shoot and root tips at the
transcriptome level. Notably, the spatial distribution and temporal ordering of the individual cells
at different developmental stages enable us to reveal continuous differentiation trajectories of
diverse plant tissues. Thus, considering the low percentage of progenitor cells in an explant,
profiling of gene expression at the single-cell level would shed light on the cell heterogeneity of
explants and help us identify the progenitor cells for SE in the future. In particular, comparisons
of explants of different origins and species may inform us whether there is a common principle
behind totipotency in plants and whether the somatic embryos are truly derived from the differen-
tiated somatic cells of explants.

Towards the epigenetic landscape of the totipotent cell state

Although ChIP-seq and ATAC-seq have been employed to dissect the epigenetic landscape
underlying SE [27], the precise molecular features of the totipotent cell state remain unclear.
First and foremost, as mentioned earlier, the progenitor cells for SE have not yet been identified.
Thus, due to the loss of information caused by signal averaging in bulk ChIP-seq and ATAC-
seq technologies, we cannot infer with certainty the chromatin-state dynamics associated
with cell reprogramming, especially during the somatic-to-totipotent transition of the progeni-
tor cells. Second, while the permissive environment (accessible to TFs and with low levels of
H3K27me3) at totipotency-related TF gene loci is a prerequisite for SE (Figure 2), it remains un-
known whether this epigenetic status is sufficient to induce somatic embryo formation. Third, al-
though zygotic and somatic embryos follow similar developmental programs after the globular
stage, it is unclear to what extent the progenitor cells of SE resemble the zygote at the chromatin
level. Last, both LEC2 and BBM are expressed from the zygote stage to the globular stage
throughout the embryo, and their expression becomes restricted at later developmental stages
[61]. Interestingly, young arabidopsis embryos are poor explants for SE [11], suggesting that the
transcriptional competence of the totipotency-related TF genes accounts for only one aspect of
SE competence.

To address these questions, the development and application of single-cell multiomics technologies
(i.e., simultaneous examination of mRNA and DNA methylation, chromatin accessibility, or histone
modification in a cell) is essential. We envision that the combination of these low-input and ultrasen-
sitive methods with cell-type, marker-based cell-sorting technology would eventually help us capture
the molecular features of the totipotent state in plants [57,58] (see Outstanding questions).

Acknowledgments
We apologize to those whose work we were unable to cite owing to space constraints. The work in J-W.W.’s laboratory is
supported by grants from the National Natural Science Foundation of China (31788103; 31721001) and the Strategic Priority
Research Program of the Chinese Academy of Sciences (XDB27030101).

Declaration of interests
No interests are declared.

Trends in Plant Science, Month 2022, Vol. xx, No. xx 7


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