AGL15 Promotion of Somatic Embryogenesis: Role and Molecular Mechanism

Abstract

Plants have amazing regenerative properties with single somatic cells, or groups of cells able to give rise to fully formed plants. One means of regeneration is somatic embryogenesis, by which an embryonic structure is formed that “converts” into a plantlet. Somatic embryogenesis has been used as a model for zygotic processes that are buried within layers of maternal tissues. Understanding mechanisms of somatic embryo induction and development are important as a more accessible model for seed development. We rely on seed development not only for most of our caloric intake, but also as a delivery system for engineered crops to meet agricultural challenges. Regeneration of transformed cells is needed for this applied work as well as basic research to understand gene function. Here we focus on a MADS-domain transcription factor, AGAMOUS-Like15 (AGL15) that shows a positive correlation between accumulation levels and capacity for somatic embryogenesis. We relate AGL15 function to other transcription factors, hormones, and epigenetic modifiers involved in somatic embryo development.

Full text

SYSTEMATIC REVIEW
published: 28 March 2022
doi: 10.3389/fpls.2022.861556
Frontiers in Plant Science | www.frontiersin.org 1March 2022 | Volume 13 | Article 861556
Edited by:
Huihui Guo,
Shandong Agricultural
University, China
Reviewed by:
Wei Gao,
Henan University, China
Xiyan Yang,
Huazhong Agricultural
University, China
*Correspondence:
Sharyn E. Perry
sperr2@uky.edu
Specialty section:
This article was submitted to
Plant Cell Biology,
a section of the journal
Frontiers in Plant Science
Received: 24 January 2022
Accepted: 01 March 2022
Published: 28 March 2022
Citation:
Joshi S, Paul P, Hartman JM and
Perry SE (2022) AGL15 Promotion of
Somatic Embryogenesis: Role and
Molecular Mechanism.
Front. Plant Sci. 13:861556.
doi: 10.3389/fpls.2022.861556
AGL15 Promotion of Somatic
Embryogenesis: Role and Molecular
Mechanism
Sanjay Joshi 1, Priyanka Paul 2, Jeanne M. Hartman 1and Sharyn E. Perry 1
*
1Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, United States, 2Kentucky Tobacco Research
and Development Center, University of Kentucky, Lexington, KY, United States
Plants have amazing regenerative properties with single somatic cells, or groups of
cells able to give rise to fully formed plants. One means of regeneration is somatic
embryogenesis, by which an embryonic structure is formed that “converts” into a
plantlet. Somatic embryogenesis has been used as a model for zygotic processes that
are buried within layers of maternal tissues. Understanding mechanisms of somatic
embryo induction and development are important as a more accessible model for
seed development. We rely on seed development not only for most of our caloric
intake, but also as a delivery system for engineered crops to meet agricultural
challenges. Regeneration of transformed cells is needed for this applied work as well
as basic research to understand gene function. Here we focus on a MADS-domain
transcription factor, AGAMOUS-Like15 (AGL15) that shows a positive correlation
between accumulation levels and capacity for somatic embryogenesis. We relate AGL15
function to other transcription factors, hormones, and epigenetic modifiers involved in
somatic embryo development.
Keywords: somatic embryo, regeneration, MADS-box, transcription factor, chromatin immunoprecipitation,
transcriptome, Arabidopsis thaliana
INTRODUCTION
Somatic embryogenesis (SE) is a powerful resource and biotechnological tool used for plant
improvement, micropropagation, and clonal regeneration (Guan et al., 2016; Ochoa-Alejo and
Loyola-Vargas, 2016). Because zygotic embryos (ZE) are embedded deep in maternal tissues, SE has
become a widely used technique to understand processes that occur during zygotic embryogenesis.
Somatic cells that undergo reprogramming to induce embryonic development produce structures
called somatic embryos.
Understanding SE is important for several other basic and applied scientific approaches that are
distinct from using SE as a model for ZE. Earth is in jeopardy due to climate change. Not only will
crops suffer from abiotic stresses such as drought and heat, but also “new” biotic stresses that can
dominate new territories due to the altered climate. While one hopes we can deal with this problem

Joshi et al. AGL15 and SE
at the source, it seems increasingly likely we will need alternative
strategies to deal with the evolving climate. One such strategy
to continue to feed our growing population is biotechnology to
engineer crops to cope with different environmental stressors
and to propagate the most valuable genotypes as efficiently as
possible (Ikeda and Kamada, 2005; Tian et al., 2020a). One
challenge to genetic engineering is the introduction of the desired
modifications into the genome. Another is the regeneration
of a plant from the modified cell(s), and SE is one means
to accomplish this latter task, although many plants and even
particular genotypes do not regenerate well by SE. SE is of
particular interest in plant developmental biology because of
its involvement in plant plasticity, especially controlling the
totipotency and pluripotency in somatic cells (Ikeuchi et al., 2016;
Fehér, 2019). Interestingly, there are some parallels with animal
cells acquiring the ability to dedifferentiate in response to stress
(Grafi, 2009).
Several studies have been performed to decipher the
regulatory mechanism of SE, and the model system Arabidopsis
thaliana (Arabidopsis) has been used for much of this work
(Yang and Zhang, 2010). Arabidopsis is a model plant due to
its available genetic resources, with a small genome size and
whole-genome sequence available from an early date (Kaul
et al., 2000). These facts and its ability to be transformed
very easily compared to other species that require intensive
tissue culture (Clough and Bent, 1998) means that there is a
database of mutants with T-DNAs inserted into nearly every
gene-of-interest, generating a loss-of-function mutant that
facilitates understanding gene function (https://conf.arabidopsis.
org/display/COM/Mutant$+$and$+$Mapping$+$Resources).
Arabidopsis zygotic embryogenesis has a predictable pattern
and basic body plan from early division (Palovaara et al., 2016;
Plotnikova et al., 2019), and SE generally follows the same stages.
While there are differences in molecular profiles between ZE and
SE, there are also congruencies worthy of further investigation
(Tian et al., 2020a). Like ZE, SE generates bipolar structures
from somatic cells with no vascular connection to the explant
tissue (Thorpe and Stasolla, 2001; Von Arnold et al., 2002). A
plethora of literature discusses genes encoding transcription
factors (TF) that are involved in seed development and can
promote SE or embryo-programs when ectopically expressed,
including the so-called LAFL factors (LEAFY COTYLEDON1
[LEC1], LEAFY COTYLEDON2 [LEC2], FUSCA3 [FUS3]
and ABSCISIC ACID INDPENDENT3 [ABI3]) (Wójcikowska
et al., 2013), BABY BOOM (BBM) (Boutilier et al., 2002; Jha
and Kumar, 2018), WUSCHEL (WUS) (Jha et al., 2020), and
AGAMOUS-LIKE15 (AGL15) (Harding et al., 2003; Zheng
et al., 2013a,b). Here we focus on AGL15 but include some
discussion of relation to the other TFs as they interact in a
complex network.
AGL15 is a member of the MADS domain TFs (named
for MCM1 from Saccharomyces cerevisiae,AGAMOUS from
Arabidopsis, DEFICIENS from Antirrhinum majus,SRF from
Homo sapiens) (Gramzow and Theissen, 2010; Chen et al.,
2017) that is expressed and primarily accumulates during early
stages of seed development, and mostly within the embryo
(Heck et al., 1995; Perry et al., 1996). The transcript level of
AGL15 in Brassica napus, and Arabidopsis, remains high during
embryo morphogenesis until the seeds start to dry (Perry et al.,
1996). Ectopic constitutive expression of AGL15 promotes SE in
Arabidopsis, Glycine max (soybean), and Gossypium hirsutum
(cotton), while mutants (loss-of-function) of both AGL15 and
AGL18 (closely related to AGL15) significantly reduce SE in
Arabidopsis (Harding et al., 2003; Thakare et al., 2008; Zheng and
Perry, 2014). Like AGL15,AGL18 is able to promote SE when
ectopically expressed in soybean and Arabidopsis (Zheng and
Perry, 2014; Paul et al., 2021).
Here we focus on genes directly regulated by AGL15 to
hypothesize about the stage of SE impacted by overexpression
of AGL15. We highlight how AGL15 takes part in different
processes involved in expression of genes to promote
somatic embryogenesis.
STAGES OF SOMATIC EMBRYOGENESIS
(SE)
In SE, the process initiates when somatic cells are induced to
change fate and form embryogenic cells that can eventually
regenerate into a complete plant. This process can be direct or
indirect (Sharp, 1980) with the latter including a callus phase.
Many factors influence the ability to undergo SE, including
plant type and even particular cultivars within a species,
physiological status of the donor plant, tissue source and age
of the explant, media components, and a variety of stresses
(Yang and Zhang, 2010).
Recent reviews have addressed the terms dedifferentiation vs.
trans-differentiation. While dedifferentiation has been used to
describe the switch from a somatic cell to an “undifferentiated”
callus, it is increasingly recognized that there are no truly
undifferentiated cells in plant regenerative processes. In fact,
callus was recently recognized as having molecular profiles
similar to root meristem tissue (Sugimoto et al., 2010).
Dedifferentiation is still used to refer to a decrease in
specialization, whereas trans-differentiation refers to a change
from one cell type to a different cell type (Fehér, 2019; Bidabadi
and Jain, 2020). While particulars to induce SE vary, a “theme”
to induce SE is the treatment of explants with auxin, usually
2,4-D, a synthetic auxin that may act as an auxin, a stressing
reagent, and/or to induce endogenous auxin production. Stress
can also take the form of wounding, temperature, and nutritional
stresses, among others. This process is thought to promote
developmental switching via a series of molecular events and
signal cascades resulting in the transition of somatic cells to
embryo identity, or callus for indirect systems (Yang and Zhang,
2010; Fehér, 2015). The unique developmental process can
be distinguished into two phases: the initiation/induction step
and the developmental phase. Cell proliferation and increased
plasticity of somatic cells occur in the first step, whereas cells
differentiate to form somatic embryos when the culture cells get
the right stimuli during the developmental phase (Magnani et al.,
2017). A brief overview of different steps follows and is shown in
Figure 1.
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Joshi et al. AGL15 and SE
FIGURE 1 | Diagram of stages of SE. Please see the text for a description of
what the numbers mean.
1. Initiation/induction—This is the step where the establishment
of pre-embryogenic and embryogenic cells or groups of cells
from the primary explant takes place. In this stage, plant
growth hormones like auxin, and in some systems, cytokinin,
play a vital role in generating cells responsive for the transition.
Stresses are also commonly applied. Large epigenetic changes
occur in response to these stimuli, and these can be seen at
a cellular level by increased nuclear volume. The competent
cells start activating the genes responsible for producing
embryogenic cells (Su et al., 2021), including genes encoding
TFs that then activate downstream pathways to generate the
SE (Quiroz-Figueroa et al., 2006).
2. Proliferation/maintenance/multiplication—Once the cells
are responsive and have entered embryogenetic fate, the
proliferation of cells initiates, which can be bulked up usually
on semisolid media or solid supports. Maintaining physical
and chemical conditions, are crucial to maintaining the
embryogenic process.
3. Maturation (histodifferentiation)—This involves
morphological stages of somatic embryogenesis analogous
to zygotic embryo development including establishment of
shoot and root apical meristems. During this step, auxin is
often removed from the media. For instance, often in indirect
systems, SEs are only obvious upon removal or a decrease
in the concentration of plant growth regulators (PGR). It is
thought molecular patterns supporting development to the
globular stage are established on the high PGR medium, and
the decrease in exogenously added PGRs is needed to continue
development (Zimmerman, 1993).
4. Post-maturation (drying, desiccation)—This stage involves
mild drying that can switch the embryo from development to
germination or for storage purposes.
5. In vitro germination (radicle elongation).
6. Conversion (establishment of the new plant).
SYSTEMS WHERE AGL15 HAS BEEN
FOUND TO PROMOTE SOMATIC
EMBRYOGENESIS (SE)
Ectopic overexpression of AGL15 has been shown to promote SE
in several plants (Figure 2). First, a system where Arabidopsis
zygotic embryo explants are placed onto Murashige and Skoog
(MS) medium lacking any exogenous plant growth regulators
(PGR) show a positive correlation between AGL15 accumulation
and ability to produce SE and maintain this development for
long time periods (Harding et al., 2003). We refer to this tissue
as Embryo Culture Tissue (ECT). Little to no apparent callus
is observed in this system. While the Arabidopsis ecotypes
Columbia (Col) and Wassilewskija (WS) wild-type (wt) embryos
can transiently produce SE tissue in this system, they switch to
leaves early. Meanwhile 35S:AGL15 increases production of ECT
in WS and leads to long term maintenance for both ecotypes
(25 years to date for WS; 10 years to date for Col) as SE tissue.
Loss-of-function or dominant negative forms of AGL15 reduce
initiation of this tissue. Because AGL15 transcript accumulation
is induced in response to auxin treatment (Zhu and Perry, 2005),
one possibility is the overexpression of transgene removes the
need for exogenous auxin. Kurczynska et al. (2007) demonstrated
that the cells competent for SE are in the protodermis and
subprotodermis of the adaxial side of the cotyledons. Whether
the 35S:AGL15 transgene enhances this competence or expands
it to other tissues remains a question for the future.
Apart from ECT, there has been another SE system where
the mature seeds are allowed to complete germination in
liquid media where synthetic auxin 2,4-D is present. After 21
days of culture, development at the shoot apex region of the
seedlings is counted for those with SE tissue and those lacking
this tissue. Other than SE development at the meristem, the
remainder of the “seedling” is callused. We refer to this system as
Shoot Apical Meristem Somatic Embryogenesis (SAM SE). The
findings show that 35S:AGL15 produced approximately twice
the frequency of seedlings with SAM SE compared to Col, wt
(for example, 56% for 35S:AGL15, compared to 29% for Col,
wt; Zheng et al., 2009). In SAM SE, 2,4-D is required to obtain
SE development; medium lacking 2,4-D simply produces liquid
grown seedlings. However, different sensitivity to 2,4-D was
observed for 35S:AGL15 compared to Col, wt (Zheng et al., 2016).
We expanded this work to Glycine max (soybean). Genes
encoding orthologs of AGL15 from soybean (GmAGL15) and
the closely related MADS-factor that has redundant functions
(GmAGL18) were cloned and introduced into SE tissue by
biolistic transformation. We found that increased expression
of these genes via a 35S promoter could 1. Enhance recovery
of transgenic lines, presumably by promoting regeneration of
transformed cells by SE and 2. Produce transgenic plants that
have enhanced ability to form SE tissue. Meanwhile, Yang
et al. (2014) cloned three AGL15 orthologs from cotton and
demonstrated that overexpression could promote SE in this plant.
While this summarizes the studies where AGL15
accumulation has been manipulated to show effects on SE
development, other studies show a correlation between SE
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Joshi et al. AGL15 and SE
FIGURE 2 | Systems where manipulation of AGL15 levels have impacted on SE. Modified from Tian et al. (2020a).(A,B) two systems in Arabidopsis; (C) in soybean;
(D) in cotton. D40, medium with 40 mg/L 2,4-D, D20, medium with 20 mg/L 2,4-D, CIM, callus induction medium.
and AGL15 accumulation. Potential orthologs of AGL15 are
shown in Figure 3.Perry et al. (1999) demonstrated that
embryos from different sources, including apomictic, microspore
embryos, and SE accumulated protein that reacted with AGL15
antiserum. Tvorogova et al. (2019) overexpressed another TF
called MtWOX9 in Medicago truncatula and found increased
SE capacity. MtAGL15 transcript accumulation increased in
response to MtWOX9. In addition, a highly embryogenic line
called 2HA showed increased MtAGL15 transcript with SE
formation. The non-embryogenic line (A17) showed no such
increase. Ghadirzadeh-Khorzoghi et al. (2019) showed increased
PvAGL15 transcript in embryogenic explants of pistachio, while
Xu et al. (2018) showed RcAGL15 associated with protocorm-like
bodies in rose.
GENE REGULATION BY AGL15 DURING
SOMATIC EMBRYOGENESIS
AGL15 is a central regulator in SE and to understand how
it directly and indirectly controls this process, expressed
and repressed target genes were identified using chromatin
immunoprecipitation (ChIP) and transcriptome studies in
Arabidopsis and soybean (Zheng et al., 2009; Zheng and Perry,
2014). Direct targets (Arabidopsis where a genome-wide study
was performed) were found to be enriched for a cis motif called a
CArG motif that is a binding site for MADS-factors like AGL15.
The consensus CArG is CC(A/T)6GG, but AGL15 can also bind
variants of this motif with a longer A/T rich core (Tang and
Perry, 2003). Studies to determine how ectopic expression of TFs
promote SE have resulted in strategies to enhance SE from non-
transgenic tissue. Examples for AGL15 include manipulation of
gibberellin metabolism and ethylene biosynthesis and response
(discussed further below).
AGL15 interacts in a complex network with several other TFs
that promote SE, including members of the LAFL TFs, BBM, and
WUS. This was recently reviewed in Tian et al. (2020a), and some
of these interactions are highlighted in Figure 4.
Comparison of targets of AGL15 in Arabidopsis and soybean
revealed some intriguing similarities. In both species, genes
involved in stress responses were overrepresented among the
direct responsive targets. These include a set of WRKY, NAC,
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Joshi et al. AGL15 and SE
FIGURE 3 | Phylogenetic tree of selected potential AGL15 orthologs, some of which promote SE development (please see text). Protein sequences were retrieved
from Phytozome (Goodstein et al., 2012) and submitted to the phylogenetic analysis tool NGPhylogeny.fr (Lemoine et al., 2019) with default settings. The output tree
was then uploaded to iTOL (version 6.5) (Letunic and Bork, 2019) for tree visualization. At, Arabidopsis thaliana; Os, Oryza sativa; GRMZM, Zea mays; Ca, Coffea
arabica; Sl, Solanum lycopersicum; Glyma, Glycine max; Mt, Medicago truncatula; Rc, Rosa canina; Pv, Pistacia vera; Gohir, Gossypium hirsutum.
and bZIP TFs that have been associated with dedifferentiation
(Grafi et al., 2011). A more recent ChIP-seq study for Arabidopsis
AGL15 revealed that regulatory regions corresponding to genes
encoding nearly all of these TFs are directly bound by AGL15,
which seems quite extraordinary (Paul et al., 2021). When
comparing the percentage of genes encoding these TFs bound by
AGL15 to the percentage of genes bound overall compared to the
whole genome, a significant enrichment in the dedifferentiation-
related (DD) TFs potentially directly regulated by AGL15 is
observed (Table 1). Interestingly, many of the other TFs that
can promote SE also show significant enrichment of these genes
among their direct targets (Table 1). Detailed information on
what SE TF associates with what DD TF gene is provided in
Supplementary Table 1.
A number of gene ontology (GO) categories are
overrepresented among the direct (as determined by ChIP-
chip and/or ChIP-seq) AGL15 targets that are expressed or
repressed. Select categories are shown in Figure 5. Notably,
genes involved in abiotic stress response are overrepresented,
as are hormone response, particularly hormones involved in
stress. Stresses such as those shown in Figure 5 are often used
to induce SE. Interestingly, genes associated with “chitin”
are overrepresented among AGL15 direct expressed targets
(Figure 5A). Several studies have found that lipophilic chitin
oligosaccharides, endochitinases, and arabinogalactan proteins
(AGP) have roles in stimulating SE (Van Hengel et al., 1998, 2002;
Domon et al., 2000) and also are responsive to stress (reviewed
in Grover, 2012; Mareri et al., 2019). One well-studied SE-related
chitinase is EXTRACELLULAR PROTEIN3 (EP3) from Daucus
carota (carrot), where addition of EP3 can rescue the production
of SE in a SE-defective line of carrot (Van Hengel et al., 1998). In
soybean, 35S:GmAGL15 was found to initially increase transcript
accumulation from a gene encoding a potential EP3 ortholog
in the explants. However, at later time points after culture on
D40 medium, differential transcript accumulation compared
to control was lost due to more extensive up-regulation in the
control compared to the overexpressor (Zheng and Perry, 2014).
DcEP3 is not expressed in the somatic (or zygotic) embryos, but
rather in cells surrounding the embryos and are thought to have
a nursing function, in part by cleavage of AGPs, some of which
have stimulatory and others inhibitory effects on SE (Toonen
et al., 1997; Van Hengel et al., 2001). Some AGPs have been
found to be important in the conditioned medium to promote
SE, and at least some are expressed in the non-embryogenic cells
in embryogenic cultures (reviewed in Von Arnold et al., 2002).
Interestingly, AGPs, including the subgroup of fasciclin-like
AGPs, (FLAs) were prevalent on the AGL15 direct repressed
list (Zheng et al., 2009), a list that became longer with the
ChIP-seq data (Paul et al., 2021) (Table 2). Only one (AGP14)
showed direct expression in response to AGL15 accumulation,
with one additional (FLA10) showing reduced transcript in
both comparisons.
AGL15 AND HORMONE RELATIONS IN
SOMATIC EMBRYOGENESIS
Hormones/PGRs play a crucial role in SE induction. Thus,
to understand SE, it is important to understand the signaling
processes related to both the exogenous and endogenous PGRs.
Generally, SE depends on the type and concentration of PGR
used for each culture medium. Recent studies showed that
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Joshi et al. AGL15 and SE
FIGURE 4 | Select interactions between TFs involved in SE and control of
hormone accumulation/signaling. Not all interactions are shown in
consideration of clarity. (A) Particular gene interactions; (B) summary of tested
or presumed AGL15 effects on different hormones. Different colored ovals with
TF names indicate different families of TFs: yellow, B3 domain; blue,
CCAAT-box binding; pale pink, APETALA2 (AP2) or AP2/ERF; mint, MADS;
dark green, BZR domain; lavender, AUX/IAA domain; orange, DELLA/GRAS;
light orange, homeodomain. Gray boxes indicate hormone or other responses.
Gray hexagon, miRNA; light blue hexagon, SERK1.
different plant species, including Arabidopsis (Wójcikowska
et al., 2013), Coffea canephora (Márquez-López et al., 2018),
and Musa spp. (Awasthi et al., 2017), successfully responded
to SE induction using variable explants, conditions, and
concentrations of PGRs. Although hormones are only part of
the composition of a culture medium, it has been estimated
that design of a new tissue culture medium requires optimizing
components that can amount to 16,000 different treatments,
leading to artificial intelligence/optimization algorithms to
assist development (Hesami et al., 2020). Thus, understanding
mechanisms of SE, including how TFs that can promote SE
regulate and respond to PGRs, is important to help guide these
efforts. Here, we focus on the relation between AGL15, select
other TFs that promote SE and select PGRs, and the underlying
mechanisms that regulate different hormone signaling pathways.
A working model is shown in Figure 4. It should be noted that
hormone interactions are exceedingly complex with cooperative
or antagonistic interactions depending on the particular context,
so this model is a simplification focusing on downstream targets
of AGL15 and how they may interact to promote SE.
AGL15 and Auxin
One of the most common components in culture media for
SE is auxin, often supplied as 2,4-D, a synthetic auxin. As
mentioned above, it has been proposed that this exogenously
added PGR may act as an auxin, as a stressing agent, and/or
to induce endogenous auxin production. Several TFs that can
induce SE respond either directly or indirectly to auxin including
BBM, FUS3, LEC1, LEC2, WUS, and AGL15 (reviewed in Tian
et al., 2020a). While it does not encode a TF per se,SOMATIC
EMBRYOGENESIS RECEPTOR LIKE KINASE 1 (SERK1), is a
key marker for cells able to form somatic embryos, and, when
expressed via a strong constitutive promoter, can enhance SE
production in multiple species. SERK1 transcript accumulation
is increased by auxin and stress (for a review, please see Mendez-
Hernandez et al., 2019). Interestingly, SERK1 has been found
to interact with protein complexes that include AGL15, as
well as proteins involved in brassinosteroid signaling (Karlova
et al., 2006 and please see below). Both SERK1 and AGL15 are
positively (possibly indirectly) regulated by WUS (Busch et al.,
2010). Further studies in Arabidopsis have demonstrated the
importance of auxin in SE (Gaj et al., 2006) as well as an optimal
2,4-D treatment that can be explained, at least in part, by auxin
effect on SE-related TF genes. All of the genes in the study showed
increased and relatively stable (over the time course) transcript
accumulation at the optimal 2,4-D concentration for SE (5 µM)
(Grzybkowska et al., 2020). However, at 3 days of culture on
various media, LEC1, LEC2 (two of the previously mentioned
LAFL factors), and BBM showed a positive correlation of
transcript accumulation over the 2,4-D concentrations used,
including sub-optimal (0.1 µM) that led to shoot development,
5µM (optimal for SE), and supra-optimal (20 µM) that produced
non-embryogenic callus, whereas AGL15 and WUSCHEL (WUS)
had very little or no transcript accumulation other than at
the SE optimal 2,4-D concentration. Grzybkowska et al. (2020)
further linked this response to epigenetic regulation (discussed
below). While AGL15 is responsive to auxin (both 2,4-D and
the biologically relevant auxin indole acetic acid, IAA), it
is not an auxin early responsive gene and requires one to
two-day treatment to see a clear response (Zhu and Perry,
2005). Consistent with this is the lack of an auxin response
element in the promoter region; but auxin may act via an
ethylene responsive element (Grzybkowska et al., 2020). Putative
orthologs of AGL15 from cotton (Figure 3) are also significantly
induced by 2,4-D, and when ectopically expressed, enhance the
embryogenic potential of calli in 2,4-D containing medium (Yang
et al., 2014). While it is unclear that monocots have MADS-
factors that, when compared to Arabidopsis MADS, are most
similar to AGL15, in Zea mays AGL15-like gene expression
was correlated with early embryogenic culture initiation. For
two of the four genes encoding these maize AGL15-like genes,
transcript accumulation increased after placement on 2,4-D
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Joshi et al. AGL15 and SE
TABLE 1 | Overrepresentation of TFs associated with dedifferentiation (DD TFs) among the genes with regions bound by TFs involved in SE.
TF # DD TFs bound Total # of genes bound Significance References
LEC1, early SE 13 3,035 0.00010 (Pelletier et al., 2017)
LEC1, late SE 17 4,890 0.00035 (Pelletier et al., 2017)
LEC1, cotyledon 22 7,216 0.00037 (Pelletier et al., 2017)
LEC2 19 2,788 5.2E-13 (Wang et al., 2020)
FUS3 4 1,218 NS (Wang and Perry, 2013)
ABI3 14 2,510 1.5E-07 (Tian et al., 2020b)
BBM 12 Top 5,000 NS (Horstman et al., 2015)
AGL15, ChIP-chip 15 3,708 7.6E-05 (Zheng et al., 2009)
AGL15, ChIP-seq 29 9,729 2.8E-05 (Paul et al., 2021)
AGL18, ChIP-seq 4 3,446 NS (Paul et al., 2021)
Chi-square test was used to compare the number of TFs associated with dedifferentiation (DD TFs; 44 total as listed in Grafi et al., 2011) bound by select regulators (TF) of embryogenesis,
compared to the total list of genes bound for each TF, using 27,334 genes total for Arabidopsis (NCBI, accessed 11/17/2021). Significance means that within the list bound by the TF,
DD TFs are overrepresented compared to expectations based on the total # of genes bound.
containing medium (Salvo et al., 2014). One of these genes is
shown in Figure 3. However, the soybean orthologs of AGL15,
(Glyma12g17721 and Glyma11g16105) did not respond to 2,4-
D, although an AGL18 ortholog (GmAGL18) was upregulated in
response to 2,4-D (Zheng and Perry, 2014).
The role of auxin in SE induction is of particular interest.
Auxin treatment is important for embryogenesis (both somatic
and zygotic); however, too high auxin concentration may hinder
the induction of embryogenic cells in the SE culture. It has
been observed that cells that are resistant to auxin in that they
maintain cell-cell adherence and do not elongate as much, may
be the particular cells capable of developing as embryos (Emons,
1994). In the Arabidopsis auxin signal transduction pathway,
the AUXIN/INDOLE-3-ACETIC ACID (AUX/IAAs) encoded
proteins bind to the Auxin Responsive Factors (ARFs), which,
in turn, are associated with DNA via auxin response elements
on auxin-responsive genes (Piya et al., 2014). AUX/IAAs
act as repressors of ARF function by keeping ARFs from
regulating gene expression until auxin perception (involving
auxin receptors TRANSPORT INHIBITOR RESPONSE1; TIR1,
and AUXIN SIGNALING F-BOX; ABFs) leads to degradation of
the IAA protein.
While auxin is important for SE, and other TFs that can induce
SE directly upregulate auxin biosynthetic genes (e.g., members
of the YUCCA gene family are induced by LEC1 and LEC2),
overexpression of YUCCA’s alone cannot induce SE (reviewed in
Braybrook and Harada, 2008). It has been proposed that other
factors, including IAA30 (discussed below) and/or AGL15, may
be important for competency to respond to auxin. Part of this
“competency” could be limiting auxin response.
AGL15 controls several genes in a manner that would reduce
auxin response and/or accumulation. In soybean, we found
that 35S:GmAGL15 reduces endogenous IAA accumulation
compared to the wild type control in developing embryos (Zheng
et al., 2016). In Arabidopsis, 35S:AGL15 directly upregulated
the gene encoding an AUX/IAA transcriptional repressor,
IAA30, which may limit auxin responses and is important
for SE (Zheng et al., 2009). An iaa30 homozygous knockout
line in both Col, wt and 35S:AGL15 backgrounds showed a
significantly lower frequency of SAM SE development than in
the corresponding background with the wild type IAA30 gene
(Zheng et al., 2009). IAA30 is also a direct expressed target
of LEC2 (Braybrook and Harada, 2008). Aux/IAAs generally
contains four domains, including an N-terminal repression
domain (domain I), domain II that enables degradation of
Aux/IAAs via ubiquitin–proteasome pathway in response to
auxin, and domains III and IV are the protein-protein interaction
domains (Guilfoyle and Hagen, 2012). However, in Arabidopsis,
IAA30 lacks the domain II that is involved in binding auxin F-box
receptors in response to auxin perception. In addition, IAA20,
which is also a potentially upregulated target of AGL15, also
lacks domain II. As a result, unlike the canonical IAA proteins,
these Arabidopsis IAA proteins have a longer half-life (Sato and
Yamamoto, 2008) and overexpression of IAA30 may interrupt the
auxin responsiveness.
In addition (Gm)AGL15 (here (Gm)AGL15 is meant to
indicate similar observations for soybean and Arabidopsis
AGL15; AGL15 is meant to indicate the observation in
Arabidopsis to date) directly represses a gene encoding an
auxin receptor (Gm)TIR1 (Zheng et al., 2016) that facilitates
the degradation of AUX/IAA in response to auxin (Hayashi,
2012), and represses both (Gm)ARF6 and (Gm)ARF8 (Zheng
et al., 2016), which are reported as transcriptional activators in
the auxin signaling pathway (Piya et al., 2014). This regulatory
interaction appears to be direct for ARF6 in Arabidopsis with
binding by AGL15 observed in ChIP experiments (Zheng
et al., 2016). Both ARF6 and ARF8 are post-transcriptionally
controlled by miRNA167 and AGL15 upregulates one of the
genes encoding this microRNA, with evidence for a direct
interaction in Arabidopsis (Zheng et al., 2016). Loss-of-function
mutants of arf6 and the double arf6/arf8 showed a significant
increase in SAM SE (Zheng et al., 2016), but in other SE
systems (culture of immature zygotic embryos), there was a
reduction in SE efficiency or productivity for the arf6 and arf8
single mutants (Wójcikowska and Gaj, 2017). Thus, context
seems important.
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Joshi et al. AGL15 and SE
FIGURE 5 | Overrepresented GO annotations among the direct (as measured
in ChIP-chip and/or ChIP-seq) and responsive (at least a 1.5-fold change at P
<0.05). Data is from the Panther Classification system accessed July 9, 2021.
(A) Directly expressed. (B) Directly repressed.
It is important to mention here that AUX/IAAS, ARFs, and
auxin receptors are multigene family proteins. Therefore, it is
feasible that other (Gm)AGL15 regulated members may promote
auxin response via other family members. For instance, there is
data supportive for direct expression of ARF5/MONOPTEROS
(MP) by AGL15 with Zheng et al. (2009) data showing a
significant reduction in mRNA in the agl15 agl18 double mutant,
although the 35S:AGL15 did not show increased accumulation
of this transcript. ARF5, when present as a loss-of-function,
had the largest effect on SE efficiency and productivity, but
overexpressing this gene did not produce any SE for the few
explants that could be obtained; again, indicating that restrictions
on auxin response may be important for SE (Wójcikowska and
Gaj, 2017). ARF3/ETTIN is repressed by AGL15, and transcript
accumulation of this ARF is negatively correlated with time
in SE culture (Wójcikowska and Gaj, 2017). Therefore, some
auxin responsive elements behave as would be believed consistent
with SE. We comment on how auxin interacts with other select
PGRs below.
AGL15 and GA
Auxin signaling leads to GA accumulation in some tissues by
activation of GA biosynthetic genes and/or GA catabolic gene
deactivation (Weiss and Ori, 2007). Therefore, it is possible that
repression of the auxin response by 35S:AGL15 may reduce
the biologically active GA, thereby promoting SE. Our prior
work corroborated this hypothesis as we found that AGL15
directly up regulates a GA catabolism gene GA2ox6 in both
Arabidopsis and soybean (Zheng et al., 2009, 2016). A reduction
of biologically active GA was also observed in 35S:AGL15
compared with the control (Wang et al., 2004). In addition, a
GA biosynthetic gene, GA3ox2 (At1g80340) was repressed by
AGL15 (Zheng et al., 2009). The accumulation of biologically
active GA was inversely correlated with SE in both Arabidopsis
and soybean (Wang et al., 2004; Zheng et al., 2016). The
addition of a GA biosynthesis inhibitor, paclobutrazol, to the
D40 medium promotes SE significantly in soybean (Zheng et al.,
2016), as well as SAM SE in Arabidopsis (Wang et al., 2004).
Interestingly, GA20ox1 (At4g25420), a gene encoding a GA
biosynthetic enzyme (Oh et al., 2014), was induced in auxin
response and directly correlated with ARF6. Thus, AGL15 also
regulates GA accumulation by controlling auxin response. In
addition, destabilization of DELLAs occurs in response to GA
(Weiss and Ori, 2007). The gibberellin-inactivated REPRESSOR
OF GA1-3 (RGA), a DELLA regulatory protein, prevents
the binding of ARF6 to DNA. GA perception leads to the
degradation of RGA, thereby enabling ARF6 to bind target genes
and cause auxin-responsive gene regulation. Another DELLA
protein, GIBBERELLIC ACID INSENSITIVE (GAI), is a putative
direct expressed target of ARF6 (Oh et al., 2014). Based on
microarray results, both the DELLA encoding genes GmRGA
and GmGAI showed increased transcript accumulation in the
35Spro:GmAGL15 on 2,4-D (Zheng et al., 2013a,b). Therefore, we
found a complex and interesting interaction between hormones
that would also include feedback mechanisms.
AGL15 and Ethylene
The effects of the gaseous hormone ethylene on plants have been
well-studied on plant growth, development, and stress responses.
The interaction between different hormonal and developmental
signals is critical in this response. According to our prior work,
(Gm)AGL15 impacts ethylene biosynthesis and perception,
which is relevant to SE (Zheng et al., 2013a). Auxin-ethylene
crosstalk reveals that they interact in a complex coregulatory
manner (Stepanova et al., 2008; Vandenbussche et al., 2012).
Auxin and ethylene impact each other’s accumulation with 2,4-
D treatment leading to an increase in ethylene accumulation
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Joshi et al. AGL15 and SE
TABLE 2 | AGP and FLA genes that show transcript response to AGL15 and are direct targets as determined by ChIP-chip or ChIP-seq.
AGI TAIR10 ChIP-chip ChIP-seq agl15agl18/Col 35S:AGL15/Col
fold-change P-value fold-change P-value
At1g03870 FLA9 Y Y 2.26 0.03 0.41 0.16
At1g35230 AGP5 Y Y 2.60 0.00 1.01 0.96
At2g04780 FLA7 Y 2.12 0.01 0.89 0.58
At2g14890 AGP9 Y Y 1.82 0.01 0.81 0.50
At2g23130 AGP17 Y Y 2.62 0.01 0.50 0.26
At2g45470 FLA8 Y Y 2.56 0.01 0.57 0.29
At2g46330 AGP16 Y 1.79 0.00 1.13 0.26
At3g06360 AGP27 Y 0.82 0.28 0.65 0.05
At3g52370 FLA15 Y Y 0.46 0.00 0.49 0.01
At3g60900 FLA10 Y 2.20 0.00 0.76 0.44
At4g09030 AGP10 Y 1.98 0.01 0.75 0.42
At4g12730 FLA2 Y 2.05 0.02 0.39 0.10
At4g37450 AGP18 Y Y 1.66 0.02 0.41 0.06
At5g11740 AGP15 Y Y 1.60 0.01 1.06 0.73
At5g56540 AGP14 Y 0.55 0.00 1.28 0.13
At5g64310 AGP1 Y 2.06 0.00 1.15 0.68
Chip-chip is from Zheng et al. (2009), but allowing transcript change of 0.67–1.5. ChIP-seq is from Paul et al. (2021), analyzed by CLC Genomics Workbench, but also including the
three sets of data analyzed by CisGenome (Ji et al., 2008). The transcriptomic data is from Zheng et al. (2009).
(Raghavan et al., 2006; Stepanova et al., 2008; Vandenbussche
et al., 2012). Conversely, TRYPTOPHAN AMINOTRANSFERASE
RELATED2 (TAR2), that encodes an αclass of pyridoxal-50-
phosphate (PLP) dependent enzymes (Stepanova et al., 2008),
was a potential direct target of AGL15 and is expressed in
response to AGL15 (Zheng et al., 2009). TAR2 is involved in
ethylene response and encodes an enzyme involved in auxin
biosynthesis (Ma et al., 2014).
In soybean, GmAGL15 was found to increase transcript
accumulation from several ethylene biosynthetic genes
and 35S:GmAGL15 tissue has significantly higher ethylene
accumulation than the control. Genes involved in ethylene
signaling were often regulated in a consistent manner in
response to AGL15 in soybean and Arabidopsis (Zheng et al.,
2013a). For example, genes encoding potential orthologs of
MtSERF1 in Arabidopsis (one gene; At5g61590) and soybean
(two genes; Glyma20g16920 and Glyma10g24360), were found
to be directly upregulated targets of (Gm)AGL15. MtSERF1 is
aMedicago truncatula ethylene response factor subfamily B-3
of APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF)
transcription factors that was shown to be required for
somatic embryogenesis. This gene is induced by ethylene and
responds to auxin and cytokinin (Mantiri et al., 2008). The
Arabidopsis ortholog of MtSERF1 was further demonstrated
to be relevant for SAM SE (Zheng et al., 2013a). These
studies allowed non-transgenic approaches to facilitate SE
in both Arabidopsis and soybean by manipulating ethylene
biosynthesis and perception. A precursor to ethylene, 1-
aminocyclopropane-1-carboxylic-acid (ACC), enhanced SE
in both species, whereas inhibitors of ethylene synthesis or
perception decreased SE.
Depending on the context, the ethylene-GA crosstalk
may be synergistic or antagonistic (Weiss and Ori, 2007).
Interestingly, transcript accumulation of both the soybean
and Arabidopsis SERF1 showed inverse correlation with GA,
supporting an antagonistic interaction in SE. In addition,
antagonistic interaction may also act by via stabilizing the
GA repressor DELLA proteins by ethylene signaling via EIN3
and CTR1 (Zheng et al., 2013a). Finally, Saptari and Susila
(2019) demonstrated that AGL15, FUS3, and BBM show
increased transcript accumulation in response to ethylene
via EIN3, whereas Zheng et al. (2013a), showed a positive
correlation between ethylene and GmAGL15, GmAGL18,
GmFUS3, and GmABI3 in soybean. Conversely, these genes
showed a general negative correlation with biologically active GA
(Zheng et al., 2016).
AGL15 and Brassinosteroids
While Brassinosteroids (BR) have been reported to enhance
SE (at least in combination with cytokinin: CK—Chone et al.,
2018), a recent publication reports on direct interactions between
AGL15, BR signaling, and SE (Ruan et al., 2021). This paper
documents experiments showing that BR promotes the embryo
to seedling transition, at least in part through repression of
AGL15 expression. Two positive regulators of BR signaling,
BRI1-EMS-SUPPRESSOR 1 (BES1), and BRASSINAZOLE-
RESISTANT 1 (BZR1), were found to bind directly to AGL15’s
promoter region. Furthermore, inhibition of BR biosynthesis
chemically or through mutants can promote SE development on
seedlings of the single mutant val1 that normally only shows
this phenotype in combination with val2 mutants (discussed
below), whereas dominant forms of bes1-D and bzr1-1D can
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Joshi et al. AGL15 and SE
rescue normal seedling development. AGL15 is involved in the
production of the SEs as shown by double and triple mutants with
an agl15 loss-of-function mutant. Ruan et al. (2021) hypothesized
that BES1 and BZR1 may also directly regulate GA biosynthetic
genes, increasing bioactive GA levels and thereby removing
negative interaction of BES1/BZR1 by the DELLA proteins.
Interestingly, Ruan et al. (2021) also show that wild
type seedlings treated with BR biosynthesis inhibitors have
darker green, downward curling leaves that look similar
to seedings overexpressing AGL15 (Fernandez et al., 2000).
AGL15 binds to the promoter regions of BZR1 and BES1
(Supplementary Figure 1) and at least for BZR1, represses
transcript accumulation. Thus, there may be an intriguing
feedback loop between BR signaling, AGL15, and GA to control
the transition from seed to seedling.
AGL15 DIRECTLY BINDS GENES
ENCODING MICRO-RNAS
MicroRNA (miRNA) function is critical during embryogenesis
through various cellular, physiological, and developmental
processes. MiRNAs target transcripts from genes that include
those with a regulatory role such as TFs that can mediate stress
adaptation, developmental regulation, and hormone response
(Jones-Rhoades et al., 2006; Jin et al., 2020). MiRNAs are
single-stranded RNA molecules of 21– 24 nucleotides that post-
transcriptionally regulate gene expression (Bartel, 2009; Rubio-
Somoza and Weigel, 2011). The mechanism of miRNA action
is either by targeting mRNA for cleavage or by inhibiting
translation (Yu et al., 2017). RNA cleavage is mediated by
recognizing the nearly perfect complementary sites (Llave et al.,
2002; Tang et al., 2003) and involves numerous proteins that are
associated with the RNA-induced silencing complex (RISC).
Briefly, MIR genes encoding microRNAs are transcribed
by RNA pol II forming primary miRNAs (pri-miRNAs) with
a characteristic hairpin structure that are processed by a
complex that includes DICER-LIKE1 (DCL1) to generate
a pre-miRNA. This pre-miRNA duplex structure is further
processed, producing the mature single stranded miRNA, which
is incorporated by ARGONAUTE1 (AGO1) to form an active
RISC (Baumberger and Baulcombe, 2005). This complex is
involved in the identification of the target transcripts that are
complementary to the miRNA sequence (Rhoades et al., 2002;
Kidner and Martienssen, 2005).
There are excellent resources on the prominence of
microRNAs, and their functions on plant development (Bartel,
2009; Rubio-Somoza and Weigel, 2011; Wójcikowska et al., 2020;
Gyawali et al., 2021; Ma et al., 2021). Several studies highlight
the crucial role of miRNA in plant development, especially
focusing on embryogenesis, including SE (Armenta-Medina
et al., 2017; Liu et al., 2018; Song et al., 2019; Alves et al., 2020;
Wójcikowska et al., 2020). Here, we emphasize how AGL15, in
addition to targeting protein-encoding genes, also regulates MIR
genes. Some miRNAs repress key embryo identity genes LEC2
and FUS3 that encode products involved in embryo maturation
(Willmann et al., 2011). Other miRNAs in Arabidopsis play
roles in cell-specific gene expression programs in response
to spatial and temporal signals, to prevent precocious gene
expression, and to enable pattern formation (Nodine and Bartel,
2010; Plotnikova et al., 2019). In the early eight-cell stage of
zygotic embryogenesis, DCL1 is essential for cell differentiation.
Similarly, dcl mutants in somatic explants are unable to initiate
embryogenic induction in vitro (Wójcik and Gaj, 2016). Another
large-scale study showed the dynamics and functions of miRNA
during Arabidopsis embryogenesis (Plotnikova et al., 2019).
The group applied a high-throughput sequencing technique
to profile hundreds of miRNAs and their targets throughout
embryogenesis. The findings highlight that miRNAs dynamically
cleave and repress at least 59 transcripts, including 30 encoding
transcription factors belonging to eight different families
(Plotnikova et al., 2019). Other studies demonstrated that
mutant lines of genes encoding miR160, miR170/171, or miR319
result in incorrect divisions in the embryo and have abnormal
cotyledon development, suggesting that the corresponding
miRNA activities are required for embryo morphogenesis
(Palatnik et al., 2003; Mallory et al., 2005; Liu et al., 2010;
Takanashi et al., 2018).
Szyrajew et al. (2017), examined the expression of 190
miRNA genes during Arabidopsis somatic embryogenesis and
found that 98% of the MIR genes were active during SE,
with 64% being differentially expressed during SE. Those
differentially expressed during SE included miR156, miR157,
miR159, miR160, miR164, miR166, miR169, miR319, miR390,
miR393, miR396, and miR398. These miRNAs are well known
to have functions associated with phytohormone and stress-
related response (Szyrajew et al., 2017). One example,miR393,
regulates the expression of auxin receptors during early SE.
The authors suggest that increased miR393, resulting in reduced
auxin sensitivity by post-transcriptional regulation of production
of auxin receptors, is involved in the transition of somatic cells to
embryogenic fate in Arabidopsis (Wójcikowska and Gaj, 2016).
Recently, Nowak et al. (2020) reported how AGL15 negatively
regulates the expression of miRNA biogenesis genes through
histone acetylation to control the embryogenic reprogramming
of somatic cells in Arabidopsis. While AGL15 accumulation
showed a positive correlation with transcript accumulation
of pri-miR156h, the mature miR156h accumulation did not
agree with the pri-miRNA and in fact the agl15agl18 mutant
showed increased mature miR156h (and miR156 from other
family members) compared to 35S:AGL15. This indicated a
disconnect between production of the pri- and mature miRNA
suggesting regulation of miRNA processing components. They
found a negative correlation between AGL15 accumulation
and transcript accumulation from the miRNA biogenesis genes
DCL1,SERRATE, and HUA-ENHANCER1 (HEN1). While they
did not perform ChIP for AGL15 for these genes, DCL1 had been
identified as a direct target of AGL15 (Zheng et al., 2009) and
they did document histone acetylation states in the regulatory
regions of these genes, demonstrating increased acetylation in the
agl15agl18 mutant and decreased acetylation in the 35S:AGL15
tissue compared to control for DCL1 and SERRATE. This would
be consistent with AGL15 recruitment of histone deacetylase
complexes to repress gene expression (discussed further below)
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Joshi et al. AGL15 and SE
(Hill et al., 2008). Neither SERRATE nor HEN1 were found as
targets in our ChIP studies (Zheng et al., 2009; Paul et al., 2021).
Our results found that AGL15 directly regulates miR167a,
with subsequent impacts on targets ARF6 and ARF8, with
consequences for SAM SE. These results are discussed under
the section on auxin. Here we present the set of miRNA
encoding genes with regulatory regions associated with AGL15
(Table 3). All of the miRNAs mentioned in Szyrajew et al. (2017)
have members bound by AGL15. Because the miRNAs were
not represented on the microarray, we looked at response of
targets of these miRNAs as predicted by TarDB (http://www.
biosequencing.cn/TarDB/guide/guide.html) (Liu et al., 2021).
Response of the targets and annotation is provided in
Supplementary Table 2.
AGL15 AND POTENTIAL LINKS TO
EPIGENETIC REGULATION OF SOMATIC
EMBRYOGENESIS
Epigenetic modifications are crucial in development processes.
The structure of chromatin can be accessible or repressive
due to various epigenetic mechanisms like DNA methylation,
histone modifications, and chromatin remodeling. These
epigenetic mechanisms are dynamic; involved in different
plant developmental phases, and in plant adaptation in various
environmental stresses. Epigenetic mechanisms could reprogram
cell fate and could stabilize cell identity and maintain tissues
(Inácio et al., 2018; Hajheidari et al., 2019). In vitro culture
conditions produce epigenetic variation and are critical factors
during SE (De-La-Pena et al., 2015; Kumar and Van Staden,
2017). Many of the loss-of-function mutants in Arabidopsis that
produce SE on “seedlings” are mutants defective in epigenetic
processes to support the transition from embryo to seedling
(reviewed in Tian et al., 2020a). Key embryo-related TFs are
inappropriately expressed in these mutants.
DNA Methylation
One well-studied epigenetic modification is DNA methylation
and this has been found to control gene expression essential
during early somatic embryogenesis (Stricker et al., 2017;
Wójcikowska et al., 2020). DNA methylation involves the
addition of a methyl group to the 5′position of the pyrimidine
ring of cytosine. In plants, methylation can occur in CpG islands
(Gruenbaum et al., 1981); CpHpG and CpHpHp (where H is
any nucleotide except G; (Feng et al., 2010). DNA methylation
differs at each stage of SE (De-La-Pena et al., 2015). Several
reports have indicated that embryogenic cultures/explants and
DNA methylation have an inverse relationship. For example,
early somatic cells of leaf explants in Coffea canephora show a
level of methylated cytosines of about 23.7%, but later stages have
higher levels of DNA methylation (Nic-Can et al., 2013). The
dedifferentiating Arabidopsis leaf protoplast has been reported
to have changes in DNA methylation (Avivi et al., 2004).
Auxin’s role in inducing epigenetic regulation of SE has been
recently reviewed (Wójcik et al., 2020). A recent study shows
hypermethylation in the promoters of TFs that are associated
with SE in response to auxin treatment (Grzybkowska et al.,
2020). They confirmed exogenous auxin substantially affected
both the expression and methylation patterns, resulting in
control of embryo-related targets LEC1, LEC2, BBM, WUS, and
AGL15, although AGL15 did not have auxin response elements
in the examined region, but did have ethylene response elements
(Grzybkowska et al., 2020). They propose that the methylation
sensitive ARF, ARF5/MP may be involved in this process.
Histone Modifications
Apart from DNA methylation, histone modifications have a
dynamic function that causes variation of gene expression that
is involved in SE. For example, the histone trimethylation mark
H3K27me3, generally a repressive mark, is removed from LEC1
loci, allowing the expression of this TF, and the expression
of BBM1 was related to the increase of both histone marks
H3K4me3 and H3K36me2 (generally marks associated with
increased transcription) using chromatin immunoprecipitation
(ChIP) assays (Nic-Can et al., 2013; Borg et al., 2020). Another
technique called immunolocalization was used in Brassica napus
to evaluate the levels of H3K9me2 that was low in microspores
before the induction of SE; however it showed increases during
later stages of SE. In contrast to methylation, it was observed that
the levels of acetylation of H3 and H4 (H3Ac and H4Ac) were
more abundant in microspores before SE induction, suggesting
it might increase transcriptional activity and regulate cellular
reprogramming and embryo development (Rodríguez-Sanz et al.,
2014). Similarly, in C. canephora, a decrease in the DNA
methylation level has been related to a decrease of H3K9me2 and
H3K27me3 during SE (Nic-Can et al., 2013).
One of the interesting groups of proteins related to histone
methylation marks that are involved in regulating plant cellular
reprogramming are the Polycomb-group (PcG). (reviewed in
Duarte-Aké et al., 2019). The PcG proteins are classified into
two complexes, POLYCOMB REPRESSIVE COMPLEX 1 and 2
(PRC1 and PRC2), which are involved in repression of genes
via histone methylation (Grossniklaus and Paro, 2014; Mozgová
et al., 2015). PRC2- mediated histone methylation represses
embryo maturation programs during vegetative development in
Arabidopsis, where cell fate could reset and SE could occur when
PRC2 depleted tissues are treated with hormone (Mozgová et al.,
2017). LEC1, LEC2, FUS3, AGL15, PLT, and WOXs, which have
regulatory roles in somatic cell differentiation and SE induction
are shown to be targeted by PRC2 in different plants (Ikeuchi
et al., 2015; Orłowska et al., 2017; Rose, 2019).
A recent study found that HSI2/VAL1 silences AGL15
to regulate the development transition from seed maturation
to vegetative phase in Arabidopsis (Chen et al., 2018).
VIVIPAROUS1/ABI3-LIKE (VAL1) and VAL2, are also known as
HIGH-LEVEL EXPRESSION OF SUGAR INDUCIBLE GENE2
(HSI2) and HSI2-LIKE1 (HSL1), respectively. VAL genes encode
B3 domain transcription factors similar to LEC2, FUS3, and
ABI3 (Suzuki et al., 2007), but also contain other conserved
domains including a plant homeodomain (PHD), a cysteine-and-
tryptophan residue containing (CW) domain, and an EAR motif
that is involved in recruiting histone deacetylation complexes.
These domains are involved in epigenetic regulation, and
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Joshi et al. AGL15 and SE
TABLE 3 | miRNAs that have associated regions bound by AGL15 (based on ChIP-chip and/or ChIP-seq). Included are predicted targets from TarDB (Liu et al., 2021)
and notations of involvement in SE and/or hormone function.
miRNA associated with
AGL15
Predicted targets of the miRNA that show significant response to AGL15 accumulation in
agl15 agl18 mutants and/or 35S:AGL15 10 d SAM SE (from TarDB: http://www.
biosequencing.cn/TarDB/)
Include targets involved in
hormone response; miRNA
involved in SE (Szyrajew
et al., 2017; Jin et al., 2020)
miR156 (a,c,d,e) AT1G11190,AT1G11840,AT1G48760,AT1G63080,AT2G27050,AT3G05430,
AT3G10910,AT3G17330,AT4G34990,AT4G35620,AT4G36650,AT1G71400,
AT2G34190,AT3G22160,AT5G43270,AT5G43460,AT5G50570,AT5G63850,
AT4G30790
Ethylene;Yes
miR157 (a,b,) AT3G22790,AT5G50570,AT1G30460,AT2G13290,AT4G16860,AT5G50570 Ethylene; Yes
miR158a AT1G17145,AT1G62930,AT1G65810,AT2G22300,AT2G46590,AT3G01040,
AT3G09850,AT3G12010,AT3G23690,AT4G30690, AT4G37740,AT5G35670,
AT5G38840,AT5G40340,AT5G43460,AT5G52060,AT5G53220
miR159 (a,b,) AT1G08030,AT1G21060,AT1G51780,AT3G44690,AT3G53810,AT4G12240,
AT1G04700,AT1G08030,AT1G08550,AT1G30210,AT1G35460,AT1G63130,
AT1G78610,AT3G10300,AT3G12550,AT3G28150,AT3G53160,AT4G23950,
AT5G04460, AT5G06100,AT5G09670,AT5G12100
ABA; Yes
miR160a AT1G52620,AT1G79180,AT4G18930,AT4G20430,AT5G43270 Auxin; Yes
miR164b AT1G76580,AT2G26690,AT4G28550,AT5G07680,AT5G62230 Yes
miR165a AT1G47530,AT1G58200,AT4G39150,AT5G50570,AT5G67570
miR166 (a,b,c,d,e,g) AT1G30490,AT1G61210,AT5G01030,AT1G30490,AT3G16785,AT5G01030,
AT1G30490, AT1G30490,AT1G30490,AT5G35670,AT5G65970,AT1G30490
Auxin; Yes
miR167a AT2G34530,AT3G59470,AT4G30130,AT5G02940,AT5G19950,AT5G49990 Auxin
miR168 (a,b) AT1G17760,AT2G02470,AT3G22160,AT1G48410,AT2G23130
miR169 (a,b,c,f) AT1G72830,AT2G01530,AT2G30600,AT3G27700,AT4G27710,AT4G36710,
AT5G12470,AT1G72830,AT5G38840, AT1G72830,AT5G38840,AT1G72830,
AT3G15950,AT3G61250,AT4G18890,AT5G62680
Yes
miR170 AT4G17370,AT5G16390,AT5G65970
miR172a AT1G12270,AT1G28190,AT1G71400,AT2G34190,AT2G47460,AT3G07060,
AT3G23330,AT4G16860,AT4G36650, AT5G02710,AT5G16260,AT5G17760,
AT5G63850
miR173 AT3G58780
miR319 (a,c) AT1G30210,AT1G04700,AT1G53230,AT3G06500 Yes
miR390 (a,b) AT3G04110,AT3G13430,AT5G05180,AT5G62230,AT1G55610,AT2G02470,
AT2G33730,AT5G62230
Auxin; Yes
miR393a AT1G15290,AT1G48410,AT2G46340,AT3G54280,AT4G26120 Auxin; Yes
miR394 (a,b) AT1G06580,AT1G19630,AT1G62910,AT3G07770,AT3G15840,AT3G48460,
AT3G48460,AT3G49240,AT4G20430,AT5G51100,AT5G56950,AT3G48460,
AT4G20430,AT4G20430,AT5G07870,AT5G09670,AT5G51100,AT5G56950
miR396b AT1G12820AT1G28010AT1G30210AT1G30490AT1G52500AT1G53AT1G55610,
AT1G60140,AT1G61370,AT1G62590,AT1G74790,AT1G76810,AT2G23130,
AT2G33730,AT2G40840,AT3G01370,AT3G03790,AT3G16730,AT3G16785,
AT3G17090,AT3G24310,AT3G54350,AT3G59420,AT4G19050,AT4G19440,
AT4G20440,AT4G30080,AT4G30130,AT4G32710,AT5G01030,AT5G12900,
AT5G39610,AT5G42830,AT5G46110,AT5G61440
Yes
miR398c AT1G30460, AT5G19950
miR399 (a,c) AT3G60040,AT3G62980,AT5G42880,AT1G71350,AT2G19470,AT2G33730,
AT2G33770,AT2G33770,AT3G06500,AT3G15130,AT3G59420,AT5G51220,
AT5G60890
miR400 AT1G07590,AT1G34670,AT1G48780,AT1G76870,AT2G13290,AT2G19470,
AT2G28550,AT2G43970,AT4G26680,AT5G14550,AT5G39710,AT5G41400,
AT5G53950,AT5G63490
mir403 AT4G31460,AT5G39900
miR858a AT1G50700,AT1G53230,AT1G65540,AT1G79640,AT3G10120,AT3G59470,
AT3G61570,AT5G46560,AT5G59900,AT5G66420
Frontiers in Plant Science | www.frontiersin.org 12 March 2022 | Volume 13 | Article 861556

Joshi et al. AGL15 and SE
HSI2/VAL1 recruits PRC2 components. During the transition
to seedling development, HSI2/VAL1 is required for the
transcriptional silencing of LAFL and AGL15 genes, but further
work demonstrated that while this protein interacts directly with
regulatory regions of AGL15, no such interaction was found for
the LAFL genes (Chen et al., 2018). While a binding site for B3
domains are involved in silencing AGL15, a mutation specifically
within the PHD domain interferes with H3K27me3 at AGL15 and
also prevents silencing, and this involved recruitment of PRC2
components (Veerappan et al., 2014; Chen et al., 2018).
Another mode of histone modification is acetylation where
lysine residues on the N-terminal tails of histones undergo
acetylation, which causes increased DNA accessibility for
TF binding. Histone acetyltransferases (HATs), and histone
deacetylases (HDACs) are the complexes that control the
acetylation state (Steunou et al., 2014; Lee and Grant, 2019).
HDAC6 and HDAC19 are two important deacetylases that are
involved in SE where a double knockdown generates SE on
seedlings. AGL15 has been found to recruit HDA19 via interact
with TOPLESS (TPL) and TOPLESS-RELATED PROTEIN2
(TPR2) (Causier et al., 2012), and SIN3 ASSOCIATED
POLYPEPTIDE P18 (SAP18; Hill et al., 2008).
DISCUSSION AND FUTURE DIRECTIONS
While AGL15 has a role in promoting SE, it is not to the
extent as found for some TFs, such as LEC1, LEC2, and
BBM, possibly indicating needed protein complex formation
with other proteins. However, the interactions between AGL15
and these other factors is fascinating, as is the fact that other
factors directly control AGL15 to facilitate the transition from
embryo to seedling. Many questions obviously remain. Why
does AGL15 appear to limit auxin responses and possibly
be involved in negative regulation of AGP signaling? These
observations may point to different roles of cells involved
in producing SEs. Whereas, some cells produce the somatic
embryos, others in the culture are equally important to provide
what has been referred to as “nursing” functions. Current
technologies allow marking and sorting of cells. An intriguing
avenue of investigation would be characterizing AGL15 function
in the cells that make SE compared to the support tissues. The
observations on GmEP3 at different timepoints of soybean SE
culture emphasize the need for examination of more timepoints
during the SE process. It is quite possible that genes responding
to AGL15 in 10 d SAM SE would behave very differently at
earlier timepoints. Other hormones are important for SE that
have not been examined for relation to AGL15 function. These
include BR, CK and ABA. Notably, genes in these GO categories
are overrepresented among AGL15 targets (Figure 5). Finally,
comparison of gene regulation in developmental processes in
different contexts will be important to tease out commonalities
and determine other context dependent factors. As an example,
Nowak et al. (2020) documented an inverse correlation between
AGL15 and transcripts from miRNA biogenesis genes, indicating
repression, direct or indirect of these genes by AGL15. However,
data from Zheng et al. (2009), showed decreased transcript
accumulation for DCL1, HEN1, and SERRATE in the agl15agl18
mutant (non-significant in the 35S:AGL15 compared to control),
suggesting expression of these genes. Zheng et al. (2009) used
an SAM SE system, whereas Nowak et al. (2020) used immature
zygotic embryos as explants, perhaps explaining these and
other differences.
Molecular breeding has become a significant tool to improve
crop production in a shorter time period, and efficient somatic
embryogenesis is a valuable technique in regenerating such crops.
Advances in gene editing techniques like CRISPR/Cas9 created
the potential to improve crop traits without regulatory concerns
that apply to traditional transgenics (once the Cas9 and guide
RNAs are segregated, at least in some countries; Grossman, 2019;
Eriksson et al., 2019). However, cells engineered for particular
traits must be able to exhibit pluripotency and produce a plant
to be useful. As yet, it is not well understood why some plants
regenerate well, poorly or not at all via either SE or organogenesis.
Even particular cultivars of a species can be recalcitrant to these
processes. We and others have found that AGL15 is a tool to
enhance regeneration via SE, at least in dicots. For example, plant
transformation in soybean is accelerated when overexpression of
AGL15 enhances somatic embryogenesis, which could be utilized
for producing elite cultivars. Similarly, AGL15 could provide a
vital role in scaling up production of valuable crops such as coffee
through propagation and genetic transformation (Etienne et al.,
2018; Xu et al., 2018). Other crops could benefit from examining
AGL15 expression in their species, and possibly enhancing
transformation strategies via better regeneration by SE.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included
in the article/Supplementary Material, further inquiries can be
directed to the corresponding author/s.
AUTHOR CONTRIBUTIONS
All authors contributed to writing the manuscript. All authors
contributed to the article and approved the submitted version.
FUNDING
This work was supported by the National Science Foundation
(grant no. IOS-1656380 to SP) and by the National Institute of
Food and Agriculture, U.S. Department of Agriculture, Hatch
project (SP) under accession number 1013409.
ACKNOWLEDGMENTS
We apologize to all of our colleagues who have generated
excellent data that we could not discuss or cite due to
space constraints.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fpls.2022.
861556/full#supplementary-material
Frontiers in Plant Science | www.frontiersin.org 13 March 2022 | Volume 13 | Article 861556

Joshi et al. AGL15 and SE
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