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plants
Review
Somatic Embryogenesis in Centaurium erythraea
Rafn—Current Status and Perspectives: A Review
Ana D. Simonovi´c, Milana M. Trifunovi´c-Momˇcilov * , Biljana K. Filipovi´c , Marija P. Markovi´c,
Milica D. Bogdanovi´c and Angelina R. Suboti´c
Citation: Simonovi´c, A.D.;
Trifunovi´c-Momˇcilov, M.M.;
Filipovi´c, B.K.; Markovi´c, M.P.;
Bogdanovi´c, M.D.; Suboti´c, A.R.
Somatic Embryogenesis in
Centaurium erythraea Rafn—Current
Status and Perspectives: A Review.
Plants 2021,10, 70.
https://doi.org/10.3390/plants10010070
Received: 15 December 2020
Accepted: 29 December 2020
Published: 31 December 2020
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional clai-
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Copyright: © 2020 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Department for Plant Physiology, Institute for Biological Research “Siniša Stankovi´c”—National Institute of
Republic of Serbia, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia;
ana.simonovic@ibiss.bg.ac.rs (A.D.S.); biljana.nikolic@ibiss.bg.ac.rs (B.K.F.); marija.nikolic@ibiss.bg.ac.rs (M.P.M.);
milica.bogdanovic@ibiss.bg.ac.rs (M.D.B.); heroina@ibiss.bg.ac.rs (A.R.S.)
*Correspondence: milanag@ibiss.bg.ac.rs
Abstract:
Centaurium erythraea (centaury) is a traditionally used medicinal plant, with a spectrum
of secondary metabolites with confirmed healing properties. Centaury is an emerging model in
plant developmental biology due to its vigorous regenerative potential and great developmental
plasticity when cultured
in vitro
. Hereby, we review nearly two decades of research on somatic
embryogenesis (SE) in centaury. During SE, somatic cells are induced by suitable culture conditions
to express their totipotency, acquire embryogenic characteristics, and eventually give rise to somatic
embryos. When SE is initiated from centaury root explants, the process occurs spontaneously
(on hormone-free medium), directly (without the callusing phase), and the somatic embryos are
of unicellular origin. SE from leaf explants has to be induced by plant growth regulators and is
indirect (preceded by callusing). Histological observations and culture conditions are compared
in these two systems. The changes in antioxidative enzymes were followed during SE from the
leaf explants. Special focus is given to the role of arabinogalactan proteins during SE, which were
analyzed using a variety of approaches. The newest and preliminary results, including centaury
transcriptome, novel potential SE markers, and novel types of arabinogalactan proteins, are discussed
as perspectives of centaury research.
Keywords:
antioxidative enzymes; arabinogalactan proteins; centaury; Gentianaceae;
in vitro
culture;
morphogenesis; plant growth regulators; somatic embryo; tissue culture
1. Somatic Embryogenesis: Biotechnological Exploitation of Plant Cells’ Totipotency
Plants have unique developmental plasticity, which allows their adaptation to constant
environmental changes. Plant
in vitro
culture techniques relies on this plasticity to mold
the morphogenetic paths in the desired direction. Morphogenetic processes enabling the
regeneration of the whole plant in
in vitro
tissue culture conditions are somatic embryogen-
esis (SE), organogenesis, micropropagation, androgenesis, and gynogenesis. Differentiated
somatic cells grown
in vitro
begin to divide and can regenerate the whole plant through
SE or organogenesis [
1
]. SE is the process during which somatic cells, under inductive
conditions, form embryogenic cells that undergo morphological and biochemical changes
leading to the formation of a somatic embryo [
2
]. SEis a powerful biotechnological method
for the propagation and genetic improvement of many plant species, as it enables the ob-
taining of a large number of somatic embryos, which can be further used in the production
of artificial seeds with diverse applications in biotechnology [3].
Somatic embryos can develop from a wide range of differentiated cell types, such as
ovule, embryo, root, leaf, and meristem cells, in response to different exogenous and/or
endogenous factors [
4
]. The presence and level of endogenous factors (phytohormones)
determine whether SE can occur spontaneously, on a hormone-free medium, or must
be induced by the addition of plant growth regulators (PGRs). SE can be direct (DSE),
Plants 2021,10, 70. https://doi.org/10.3390/plants10010070 https://www.mdpi.com/journal/plants
Plants 2021,10, 70 2 of 19
without an intermediate callusing phase, or indirect (ISE), which implies the formation of
disorganized callus tissue [5]. Somatic embryos developed through DSE or ISE may be of
uni- or multicellular origin [6].
Somatic cells are not totipotent per se, and they need induction under appropriate con-
ditions [
7
]. During the induction phase, somatic cells acquire embryogenic competence and
proliferate, while during the expression phase, embryogenic cells differentiate into somatic
embryos [
8
]. These two phases are thought to be mutually independent and influenced by
different factors. Competent cells represent a transition from somatic to embryogenic state,
which still requires exogenous stimuli, while the embryogenic cells have the ability to regen-
erate embryos without exogenous stimuli [
8
,
9
]. Inductive conditions, such as exogenously
added PGRs and stress factors, lead to the dedifferentiation of plant cells and activation of
the embryogenic pathway [
10
]. It is still unclear why and how differentiated plant cells
become totipotent and acquire embryogenic potential and why this phenomenon occurs
only in certain plant species, certain tissue types, or cells [
10
]. Many genes are involved
in the vegetative to embryogenic transition. These include phytohormone-responsive
genes, such as auxin-related and ABA-inducible genes, genes involved in the cell cycle
control, genes involved in growth and remodeling of the cell wall, as well as an array
of transcription factors. The involvement of LEC (leafy cotyledon), BBM (baby boom),
WUS (wuschel), CLV (clavata), STM (shoot meristemless), SHR (short root), ABI3 (abscisic
acid-insensitive), FUS3 (fusca) and other transcription factorshave been confirmed in SE
regulation [
11
]. Somatic Embryogenesis Receptor-Like Kinases (SERKs) are well-known
SE-specific signaling components [
12
]. Although significant progress in identifying factors
involved in induction, perception, and signal transduction during SE has been made,
the results of these numerous studies are still fragmentary and insufficient to explain the
events occurring during SE at the molecular level.
While the initiation of embryogenic tissues depends on the developmental stage of the
used initial plant material and components of the nutrient medium, the sustention of the
embryogenic potential during subsequent cultivation requires the simultaneous activity
of signaling and genetic pathways. Identification of proteins and genes involved in the
control of the embryogenic potential of the plant cells represents one of the most efficient
ways for understanding the molecular mechanisms of SE. These molecules, so-called SE
markers, can allow the identification of the cells with embryogenic potential in the tissue
culture before visible morphological changes. Several proteins isolated during SE are
stress-related or pathogen-related proteins. These proteins were isolated from different
plants during stress treatments of plant tissue culture imposed by wounding, desiccation,
heavy metals, or PGRs [
13
]. Extracellular proteins are also potentially good markers
of SE because they play an important role in plant cell differentiation [
14
]. The largest
number of extracellular proteins are glycoproteins, of which arabinogalactan proteins
(AGPs) are especially important during SE. The current review covers several aspects of SE,
including the influence of explant type and culture conditions (PGRs and light conditions),
as well as the roles of antioxidative enzymes and AGPs, investigated in a medicinal plant
Centaurium erythraea.
2. Centuries of Centaury Research
Centaurium erythraea Rafn, commonly known as centaury, is a pharmacologically im-
portant medicinal plant from the Gentianaceae family. Centaury is a biennial, sometimes
annual herb, which grows in wet to semi-arid areas throughout the northern hemisphere
It is an ancient medicinal plant with the longest tradition in many pharmacopeias: it was
described by Dioscorides nearly 2000 years ago. Centaury is used for the treatment of a
wide range of ailments [
15
–
17
]. Using pure phytochemicals or crude plant extracts, experi-
mental trials have been performed to evaluate anticancer, antioxidant, anti-inflammatory,
antipyretic, analgesic, antimicrobial, antidiabetic, gastro-,cardio- and hepatoprotective ac-
tivities of this important plant in many experimental animal systems [
18
–
24
]. A wide range
of bioactive compounds can be found in the aerial part of C. erythraea, including secoiri-
Plants 2021,10, 70 3 of 19
doids, indole alkaloids, phenolic compounds (xanthones, flavonoids, and phenolic acid),
and terpenes [
25
–
27
]. Centaur can also be used in the food processing industry as a natural
flavoring or additive [26].
The ever-expanding demand for centaury in traditional medicine and the pharmaceu-
tical industry has led to its uncontrolled collection resulting in a rapid decline of its natural
populations. Development of methods for
in vitro
mass propagation of the centaury plants,
as well as strategies for biotechnological production of its active metabolites, have attracted
the attention of several research groups, resulting in a number of publications.
C. erythraea is relatively easily manipulated in the
in vitro
culture, where it can even
complete its life cycle. Centaury’s manageability and developmental plasticity
in vitro
made it not only the most investigated species of the Centaurium genus but an emerging
model in plant developmental biology. The diversity of morphogenetic paths that C. ery-
thraea can undergo
in vitro
has been compiled recently [
28
]. The vigorous morphogenic
potential of the explants favors the use of centaury for genetic transformations [
29
–
31
],
interspecific hybridization
in vitro
[
32
–
34
], functional studies on secondary metabolite syn-
thesis
in vitro
[
25
,
26
,
35
–
37
], and stress physiology studies [
38
,
39
]. Overall, different aspects
of C. erythraea’s
in vitro
development, physiology, pharmacology, and ecology have been
studied for over20 years at the Department for Plant Physiology, Institute for Biological
Research “Siniša Stankovi´c”, University of Belgrade, resulting in 38 journal papers, 8 book
chapters, and 9 masters and doctoral theses. Similarities and differences between two
in vitro
systems for the induction of SE in centaury that have been extensively studied
in our lab—spontaneous DSE from root culture and induced ISE from leaf explants—are
the focus of this review. A newly developed system for secondary and cyclic SE is also
described [40] and will be submitted as an accompanying article in this issue.
3. SE from Centaury Root Explants Is Spontaneous and Direct
Both SE and organogenesis
in vitro
can proceed either spontaneously, on hormone-free
media, or be induced by (a combination of) PGRs. PGRs exogenously added to the nutrition
medium, as well as the content of endogenous hormones in different plant tissues, affect the
induction of SE [
8
,
9
,
41
]. Auxins and cytokinins (CKs) are the main factors that determine
the response and direction of SE by controlling the cell cycle, activation of cell divisions,
and cell differentiation [
5
,
8
], so their presence is required for SE induction
[42–44].
However,
only certain auxins, such as 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic
acid (NAA), were key factors for inducing embryogenic cells from immature leaves of
Manihot esculenta [
45
]. In addition, 2,4-D or picloram (PIC) induced direct SE in leaf
segments of Petiveria alliacea [
46
]. On the other hand, there are reports describing that CKs,
such as thidiazuron (TDZ) and 6-benzylaminopurine (BA), played a crucial role in SE form
leaf and shoot explants of Ochna integerrima and leaf explants of Primulina tabacum [
47
,
48
].
The first successful induction of SE in centaury was obtained in a cell suspension
derived from callus cultures [
49
]. The calli were initiated from roots and shoots of seedlings
on medium supplemented with kinetin (KIN, 10
−6
M) and auxins indole-3-acetic acid
(IAA, 10
−5
M) or 2,4-D (10
−6
M). In contrast to 2,4-D, IAA showed a stimulatory effect on
SE induction from the cell suspension, but light was the main embryogenesis-inducing
factor in this system.
Suboti´c et al. [
35
] achieved the induction of SE from centaury root culture on solid half-
strength Murashige and Skoog medium (
1
2
MS) without the addition of PGRs in the light.
The somatic embryos developed alongside adventitious buds, so somatic embryos and
adventitious buds of different developmental stages could be observed on the same root
explant. Histological studies revealed that the somatic embryos formed directly from the
epidermal cells, without the callusing phase, while adventitious buds developed from
root cortex tissue [
50
]. In other words, SE from centaury root explants was spontaneous,
direct (DSE), asynchronous, and occurred simultaneously with organogenesis. Somatic
embryos derived from root explants of
in vitro
-grown centaury followed a unicellular
pathway of DSE. These observations were in accordance with an earlier report describing
Plants 2021,10, 70 4 of 19
in vitro
morphogenesis from apical segments of primary hairy roots [
29
]. Even though
the DSE from roots occurred spontaneously, the effects of PGRs added to the culture were
further investigated. Suboti´c et al. [
51
] reported the effects of exogenous gibberellic acid
(GA
3
) and paclobutrazol, an inhibitor of gibberellin synthesis, both added at concentrations
0.01–3.0
µ
M, on the SE induction in wild type and hairy root centaury culture. It was
shown that GA
3
had an inhibitory effect on the process of SE, while paclobutrazol in all
applied concentrations had a stimulatory effect. The induction of SE in solid centaury root
culture is presented in Figure 1.
Plants 2020, 9, x FOR PEER REVIEW 4 of 20
lowed a unicellular pathway of DSE. These observations were in accordance with an
earlier report describing in vitro morphogenesis from apical segments of primary hairy
roots [29]. Even though the DSE from roots occurred spontaneously, the effects of PGRs
added to the culture were further investigated. Subotić et al. [51] reported the effects of
exogenous gibberellic acid (GA3) and paclobutrazol, an inhibitor of gibberellin synthesis,
both added at concentrations 0.01-3.0 µM, on the SE induction in wild type and hairy
root centaury culture. It was shown that GA3 had an inhibitory effect on the process of
SE, while paclobutrazol in all applied concentrations had a stimulatory effect. The in-
duction of SE in solid centaury root culture is presented in Figure 1.
Figure 1. Direct somatic embryogenesis (SE) in Centaurium erythraea solid root culture. (a) The first response of root ex-
plant is enlargement and clear morphological changes observed five days after the culture setup on half-strength Mu-
rashige and Skoog (½MS) medium, (b) Detail of root explant with somatic embryos, (c) Cotyledonary somatic embryos
developed directly from the root explant with no intervening callus phase, (d) Cross-section of root explant at the begin-
ning of the culture. Scale bar indicates 200 µm, (e) Histological appearance of a proembryogenic structure. Scale bar in-
dicates 100 µm, (f) Somatic embryo originated directly from the epidermal and subepidermal cells of the root tissue. Scale
bar indicates 100 µm.
As recently reviewed by Tomiczak et al. [52], SE has also been successfully obtained
from the root cultures of several other species from the Gentianacea family, even though
in these cases, the SE was not spontaneous as in centaury but required the addition of
PGRs. Mikuła and Rybczyńsky [53] tried to induce SE in G. cruciate root explants cul-
tured on MS medium supplemented with 2,4-D and kinetin. The root explants formed
callus tissue at the cut surface precisely on the wounding site of roots. Further ultra-
structural analysis has shown that the structures originating from single cortical cells
resembled proembryos in root explants of G. cruciate, but the process of SE was not fur-
ther continued [54]. On the other hand, in G. kurroo, G. pannonica,and G. cruciate, somatic
embryos were regenerated by rhizodermal cells of adventitious roots [43]. This process
was stimulated by various combinations of auxins and CKs, and somatic embryos were
further converted into plantlets on a ½MS medium. Successful induction of SE was also
achieved on root explants of G. lutea grown on a medium supplemented with auxins
alone or in combination with cytokinin, although the conversion of somatic embryos in-
to plantlets required the addition of mannitol or sorbitol to the basal culture medium
[55]. The initiation of somatic embryos was also obtained in root explants of Eustoma
grandiflorum cultured on a medium with 2,4-D [56]. The somatic embryos originated
from pericycle and vascular parenchyma cells of seedling roots. Further conversion of
the somatic embryos into plantlets was enabled with the addition of BA or GA3 [56].
Figure 1.
Direct somatic embryogenesis (SE) in Centaurium erythraea solid root culture. (
a
) The first response of root explant
is enlargement and clear morphological changes observed five days after the culture setup on half-strength Murashige
and Skoog (
1
2
MS) medium, (
b
) Detail of root explant with somatic embryos, (
c
) Cotyledonary somatic embryos developed
directly from the root explant with no intervening callus phase, (
d
) Cross-section of root explant at the beginning of the
culture. Scale bar indicates 200
µ
m, (
e
) Histological appearance of a proembryogenic structure. Scale bar indicates 100
µ
m,
(
f
) Somatic embryo originated directly from the epidermal and subepidermal cells of the root tissue. Scale bar indicates
100 µm.
As recently reviewed by Tomiczak et al. [
52
], SE has also been successfully obtained
from the root cultures of several other species from the Gentianacea family, even though in
these cases, the SE was not spontaneous as in centaury but required the addition of PGRs.
Mikuła and Rybczy´nsky [
53
] tried to induce SE in G. cruciate root explants cultured on MS
medium supplemented with 2,4-D and kinetin. The root explants formed callus tissue at
the cut surface precisely on the wounding site of roots. Further ultrastructural analysis has
shown that the structures originating from single cortical cells resembled proembryos in
root explants of G. cruciate, but the process of SE was not further continued [
54
]. On the
other hand, in G. kurroo,G. pannonica, and G. cruciate, somatic embryos were regenerated
by rhizodermal cells of adventitious roots [
43
]. This process was stimulated by various
combinations of auxins and CKs, and somatic embryos were further converted into plantlets
on a
1
2
MS medium. Successful induction of SE was also achieved on root explants of
G. lutea grown on a medium supplemented with auxins alone or in combination with
cytokinin, although the conversion of somatic embryos into plantlets required the addition
of mannitol or sorbitol to the basal culture medium [
55
]. The initiation of somatic embryos
was also obtained in root explants of Eustoma grandiflorum cultured on a medium with
2,4-D [
56
]. The somatic embryos originated from pericycle and vascular parenchyma cells
of seedling roots. Further conversion of the somatic embryos into plantlets was enabled
with the addition of BA or GA3[56].
Plants 2021,10, 70 5 of 19
4. Indirect SE from Centaury Leaf Explants
The successful induction of SE, as well as shoot and root regeneration
in vitro
, depends
on a variety of factors, including the explant selection, light conditions, and exogenously
added PGRs [
5
,
57
]. The leaf culture, implying
in vitro
cultivation of isolated leaves, is gen-
erally not used because the whole leaves cannot be maintained in tissue culture. However,
if the leaf sections are used as initial explants, then calli, buds, or somatic embryos can
be induced since some mesophyll cells have the potential to re-enter the cell cycle and
become committed to different morphogenetic pathways when appropriately induced.
Leaves from
in vitro
cultivated plants are an easily accessible source of explants, while the
leaf culture enables the regeneration of genetically stable plants [58].
The effect of nutrient media and different PGRs on regeneration possibilities from
centaury leaf explants have been investigated in several previous studies [
59
–
62
], but in all
these reports, only adventitious buds and calli regenerated on the leaf explants. Recent re-
search revealed that centaury leaf explants cultured on hormone-free medium in the light
produced only a few shoots, while roots developed in darkness [
28
]. Both organogenesis
and rhizogenesis occurred directly, without the callusing stage, but no somatic embryos
developed on hormone-free media [28].
The first successful induction of SE from the centaury leaf explants was reported by
Filipovi´c et al. [
28
] on media containing N-(2-chloro-4-pyridyl)-N’-phenylurea (CPPU) and
2,4-D, applied together, where the embryogenic response increased with the increasing
CPPU concentration. Synthetic urea-type cytokinin CPPU has a diverse morphogenic ac-
tivity in different species [
63
]; other tested PGRs with cytokinin activity—6-benzyladenine,
kinetin, and thidiazuron—induced callus proliferation only [
28
]. This combination of
PGRs(CPPU + 2,4-D) induced somatic embryo formation in Gentiana spp. leaf explants,
as well [
43
]. When the centaury leaf explants are cultivated on CPPU and 2,4-D, the direc-
tion of morphogenesis depends on the light conditions: If the explants are cultivated in
darkness, the indirect formation of somatic embryos (ISE) is the only process that occurs,
but when the explants are kept in the light, the processes of ISE and indirect shoot devel-
opment (ISD) proceeded simultaneously, and both were asynchronous [
28
]. Even though
ISE can be isolated from other morphogenetic paths by culturing the explants in darkness,
a higher frequency of embryogenic callus induction was obtained in the light. Thus, it can
be concluded that in centaury leaf culture, light is an obligatory factor for the organogenesis,
but also a factor that enhances ISE [
28
], which is in accordance with previous reports where
light-induced SE in centaury suspension culture [
49
], as well as the frequency of SE and the
number of embryos per leaf explant of Dendrobium [
64
] and Petiveria alliacea cultures [
46
].
The developed somatic embryos originated from the leaf subepidermal cells [28].
Plant regeneration via SE in leaf culture was also obtained in other gentian species.
ISE in G. pneumonanthe was achieved on
1
2
MS supplemented with 2,4-D and BA [
65
].
The embryogenic potential of leaf explants was also investigated in G. kurroo,G. cruciata,
G. tibetica,G. lutea,G. pannonica [
43
]. The leaf explants of these species were grown on a
medium supplemented with three auxins and five different CKs, and optimum regeneration
was achieved in the presence of NAA in combination with BA or TDZ (thidiazuron).
Furthermore, cytomorphological analyses have shown that somatic embryos originated
from palisade mesophyll cells. SE was also induced on leaf explants of G. straminea and
G. utriculosa cultured on an MS medium supplemented only with 2,4-D [
66
,
67
]. On the
other hand, in leaf explants of G. straminea,G. macrophylla, and S. chirata, successful
induction of embryogenic callus was achieved on medium with a combination of 2,4-
D and CKs
[68–70].
The process of ISE from the centaury leaf explants is illustrated in
Figure 2and the Supplementary video.
Plants 2021,10, 70 6 of 19
Plants 2020, 9, x FOR PEER REVIEW 6 of 20
Figure 2. SE in Centaurium erythraea leaf culture. (a) Embryogenic callus developed at the edge of the leaf explant treated
with 2,4-dichlorophenoxyacetic ac(2,4-D) and N-(2-chloro-4-pyridyl)-N’-phenylurea (CPPU) in darkness, (b) and(c) So-
matic embryos at different stages of development (arrows), (d-f) Micrographs showing somatic embryo development on
a leaf explant, (d) Histological appearance of a meristematic center (arrow) in the subepidermal layer of the leaf explant.
Scale barindicates 200 µm, (e) Globular somatic embryo. Scale barindicates 100 µm, (f) Cotyledonary somatic embryo
with apical meristem (AM), leaf primordial (LP), and provascular bundles (PB). Scale barindicates 200 µm.
Since the two main systems for the SE induction in centaury, DSE from root culture
[35] and ISE from leaf culture [28], differ in their requirements regarding the addition of
PGRs for the SE induction, the endogenous contents of different CKs, IAA, salicylic acid
(SA) and abscisic acid (ABA) were analyzed in the roots and shoots of the in vitro grown
plants as the sources of explants [71]. It was found that the total amount of endogenous
CKs was 1.4 times higher in shoots as compared to shoots, but inactive or weakly active
N-glucosides were the predominate CK forms in both organs, whereas free bases and
O-glucosides represented only a small portion of the total CK pool. The roots were
characterized with higher IAA content but lower IAA/free CK bases ratio and lower
ABA content in comparison to roots. The most significant difference, however, was a
44-fold higher SA content in the roots as compared to shoots [71]. It is not clear which of
these differences allows spontaneous SE from roots but not from shoots; for example,
Quiroz-Figueroa et al. [2] demonstrated that very low concentrations of salicylates could
induce cellular growth and enhance somatic embryogenesis in Coffea arabica. Planned
investigation considering the determination of endogenous levels of phytohormones at
different stages of somatic embryo development aims to relate these levels to the em-
bryogenic capacity of centaury root and shoot explants. The processes of SE from cen-
taury root and leaf cultures are presented in Figure 3.
Figure 2.
SE in Centaurium erythraea leaf culture. (
a
) Embryogenic callus developed at the edge of the leaf explant treated
with 2,4-dichlorophenoxyacetic ac(2,4-D) and N-(2-chloro-4-pyridyl)-N’-phenylurea (CPPU) in darkness, (
b
) and (
c
) Somatic
embryos at different stages of development (arrows), (
d
–
f
) Micrographs showing somatic embryo development on a leaf
explant, (
d
) Histological appearance of a meristematic center (arrow) in the subepidermal layer of the leaf explant. Scale bar
indicates 200
µ
m, (
e
) Globular somatic embryo. Scale bar indicates 100
µ
m, (
f
) Cotyledonary somatic embryo with apical
meristem (AM), leaf primordial (LP), and provascular bundles (PB). Scale bar indicates 200 µm.
Since the two main systems for the SE induction in centaury, DSE from root culture [
35
]
and ISE from leaf culture [
28
], differ in their requirements regarding the addition of PGRs
for the SE induction, the endogenous contents of different CKs, IAA, salicylic acid (SA)
and abscisic acid (ABA) were analyzed in the roots and shoots of the
in vitro
grown
plants as the sources of explants [
71
]. It was found that the total amount of endogenous
CKs was 1.4 times higher in shoots as compared to shoots, but inactive or weakly active
N-glucosides were the predominate CK forms in both organs, whereas free bases and
O-glucosides represented only a small portion of the total CK pool. The roots were
characterized with higher IAA content but lower IAA/free CK bases ratio and lower ABA
content in comparison to roots. The most significant difference, however, was a 44-fold
higher SA content in the roots as compared to shoots [
71
]. It is not clear which of these
differences allows spontaneous SE from roots but not from shoots; for example, Quiroz-
Figueroa et al. [
2
] demonstrated that very low concentrations of salicylates could induce
cellular growth and enhance somatic embryogenesis in Coffea arabica. Planned investigation
considering the determination of endogenous levels of phytohormones at different stages
of somatic embryo development aims to relate these levels to the embryogenic capacity of
centaury root and shoot explants. The processes of SE from centaury root and leaf cultures
are presented in Figure 3.
Plants 2021,10, 70 7 of 19
Plants 2020, 9, x FOR PEER REVIEW 7 of 20
Figure 3. Schematic overview of SE in Centaurium erythraea. Left panel: Both leaves and roots of the in vitro grown C. er-
ythraea plants can serve as sources of explants for the induction of SE. Hairy root cultures can also be used as explants.
Upper panel: SE can be induced from leaf explants on the inductive medium containing 2,4-D and CPPU, both in the light
and in darkness. Somatic embryos form from differentiated somatic cells in the subepidermal layer of the leaf explant. In
this case, SE is indirect and proceeds via the callusing phase. Middle panel: The obtained somatic embryos can be further
used as explants for secondary or cyclic embryogenesis [40] Lower panel: SE from root or hairy root explants is sponta-
neous, on ½ MS medium and direct. SE starts with asymmetric divisions of single totipotent cells from the epidermal or
subepidermal layers of root explant. Successive divisions give rise to somatic embryos.
5. Maintaining Reactive Oxygen Species Homeostasis during SE in Centaury: The
Role of Antioxidative Enzymes
Three decades ago, Dudits et al. [72] suggested that the somatic embryo initiation in
vitro was a stress response. Many reports since then underlined the importance of stress
factors during SE induction in vitro [10,73–76]. Cultured plant tissues experience a vari-
ety of stresses as a consequence of in vitro manipulations, including wounding, steriliza-
tion, mineral nutrient imbalance in the culture medium composition, PGRs, or subcul-
tures. In response to any of these stresses, the homeostasis between reactive oxygen spe-
cies (ROS) production and scavenging is disturbed, and ROS are generated in excess,
thereby imposing oxidative stress in plant tissue culture. Stresses experienced by cul-
tured tissues may induce a general response, resulting in chromatin remodeling and ac-
tivation of the embryogenic developmental program [72,74]. Namely, accumulating ev-
idence revealed that ROS (specifically H2O2) may function as signaling molecules that
regulate plant growth and development, including cellular proliferation and differentia-
tion [77,78]. As a cellular messenger, H2O2 in proper concentrations has the ability to in-
duce gene expression and protein synthesis, hence triggering activation of embryogenic
developmental program and formation of somatic embryos in different plant species [76].
On the other hand, excessive ROS could severely damage cellular proteins, DNA, and
membrane lipids [78]. Thus, ROS overproduction could lead to plant recalcitrance and
reduced morphogenetic competence during the in vitro culture [79]. Therefore, main-
taining an optimum ROS level in the cell and restoring cell redox balance is important
and enables the regulation of various processes [78], including SE induction.
Figure 3.
Schematic overview of SE in Centaurium erythraea. Left panel: Both leaves and roots of the
in vitro
grown
C. erythraea plants can serve as sources of explants for the induction of SE. Hairy root cultures can also be used as explants.
Upper panel: SE can be induced from leaf explants on the inductive medium containing 2,4-D and CPPU, both in the light
and in darkness. Somatic embryos form from differentiated somatic cells in the subepidermal layer of the leaf explant.
In this case, SE is indirect and proceeds via the callusing phase. Middle panel: The obtained somatic embryos can be
further used as explants for secondary or cyclic embryogenesis [
40
] Lower panel: SE from root or hairy root explants is
spontaneous, on
1
2
MS medium and direct. SE starts with asymmetric divisions of single totipotent cells from the epidermal
or subepidermal layers of root explant. Successive divisions give rise to somatic embryos.
5. Maintaining Reactive Oxygen Species Homeostasis during SE in Centaury:
The Role of Antioxidative Enzymes
Three decades ago, Dudits et al. [
72
] suggested that the somatic embryo initiation
in vitro
was a stress response. Many reports since then underlined the importance of stress
factors during SE induction
in vitro
[
10
,
73
–
76
]. Cultured plant tissues experience a variety
of stresses as a consequence of
in vitro
manipulations, including wounding, sterilization,
mineral nutrient imbalance in the culture medium composition, PGRs, or subcultures.
In response to any of these stresses, the homeostasis between reactive oxygen species (ROS)
production and scavenging is disturbed, and ROS are generated in excess, thereby impos-
ing oxidative stress in plant tissue culture. Stresses experienced by cultured tissues may
induce a general response, resulting in chromatin remodeling and activation of the em-
bryogenic developmental program [
72
,
74
]. Namely, accumulating evidence revealed that
ROS (specifically H
2
O
2
) may function as signaling molecules that regulate plant growth
and development, including cellular proliferation and differentiation [
77
,
78
]. As a cellular
messenger, H
2
O
2
in proper concentrations has the ability to induce gene expression and
protein synthesis, hence triggering activation of embryogenic developmental program and
formation of somatic embryos in different plant species [
76
]. On the other hand, excessive
ROS could severely damage cellular proteins, DNA, and membrane lipids [
78
]. Thus,
ROS overproduction could lead to plant recalcitrance and reduced morphogenetic com-
petence during the
in vitro
culture [
79
]. Therefore, maintaining an optimum ROS level in
the cell and restoring cell redox balance is important and enables the regulation of various
processes [78], including SE induction.
Plants 2021,10, 70 8 of 19
The level of H
2
O
2
is controlled by the activities of several key enzymes, including
superoxide dismutases (SODs), catalases (CATs), and class III peroxidases (POXs) [
80
].
The SODs provide the front-line defense against ROS since they scavenge superoxide radi-
cals to produce H
2
O
2
[
81
]. CATs remove the excess of H
2
O
2
, while extracellular POXs play
a role in the precise regulation of ROS levels in the cell and apoplast because, in addition to
their role in removing H
2
O
2
, they can also catalyze the formation of H
2
O
2
and hydroxyl
radicals [
82
–
84
]. By regulating ROS levels in the apoplast, POXs participate in cross-linking,
cell wall reconstruction, and elongation [
81
]. In many plants, these antioxidant enzymes
have been shown to play an important role in scavenging ROS that arise during SE [
75
,
77
].
To our best knowledge, the only study on the roles of antioxidative enzymes in relation
to SE within the Gentianaceae family is the study on the already described system of regen-
eration and ISE induction from centaury leaf explants [
28
]. Filipovi´c et al. [
28
] investigated
the activities of SODs, CATs, and POXs in a comprehensive set of samples comprising
intact leaves, wounded explants, and explants grown either in light or darkness on three
types of media, of which one inductive medium (0.2 mg/L 2,4-D and
0.5 mg/L CPPU
)
supported ISE. Of these, only the changes in the antioxidative activities in response to
wounding and during ISE will be discussed here.
Wounding of the centaury leaves (cutting the leaves into explants) caused an increase
in SOD activity (comprising 3 Cu/Zn-SOD isoforms), an increase in CAT activity (compris-
ing 3 major activity bands), as well as a decrease in total POX activity [
28
], indicating that
SOD and CAT are involved in the protection of centaury leaves from wounding-induced
oxidative damage. Wounding leads to an accumulation of ROS in Medicago truncatula
leaf explants, which occurs within seconds [
85
]. Slesak et al. [
86
] showed that mechanical
injury of Mesembryanthemum cristallinum leaves leads to H
2
O
2
accumulation, which was
accompanied by an increase in total SOD activity and a decrease in CAT activity. Decreased
POX activity in response to wounding, recorded in centaury leaves, is consistent with
low POX activity in freshly isolated leaf explants of Dactylis glomerata [
1
]. Mechanical
wounding is an inevitable consequence of
in vitro
manipulations. Wound signaling trig-
gers not only defense responses, such as the production of ROS but also healing responses,
including dedifferentiation, cell cycle reactivation, and vascular regeneration [87].
Following rapid responses to wounding, ROS homeostasis has to be reestablished,
which is crucial for initial cell dedifferentiation and division during callus formation [
88
].
Subsequent planting of the centaury leaf explants on inductive medium strongly induced
total POX activity, both in light and darkness, suggesting the importance of these enzymes
in cell division, growth, and differentiation, probably through their action on cell wall
remodeling [
82
]. A statistically significant increase in POX activity in comparison to the
control intact leaves occurred after seven days of incubation, when the first cell divisions
and the formation of meristem centers in the sections of the centaury leaves were observed,
with the peaks of POX activity on the 14th or 21st day in culture, which coincidence with
the emergence of somatic embryos. Therefore, it could be concluded that POXs play an
important role in the development of centaury somatic embryos. Previous reports con-
firmed the important role of POX during SE induction from leaf explants of D. glomerata [
1
]
and Cicer arietinum [
44
]. On the contrary, SOD activity decreased in light and remained
unchanged during ISE in darkness, while CAT activity decreased during ISE both in light
and darkness. The obtained results illustrate that dynamic changes in the antioxidative en-
zymatic capacity upon wounding and in response to SE induction are required to maintain
ROS homeostasis in centaury leaf explants.
6. Studies on the Role of AGPs during SE in Centaury Using β-D-glucosyl
Yariv Reagent
AGPs are heavily glycosylated, intrinsically disordered glycoproteins ubiquitous in
plants, which belong to a superfamily of cell surface hydroxyproline-rich glycoproteins
(HRGPs) [
89
,
90
]. The extraordinary structural diversity of AGPs relies not only on their
protein backbones encoded by large gene families [
89
,
91
] but also on the possibility of dif-
ferential glycosylation of the same isoform into hetero generous glycoforms [
92
]. Structural
Plants 2021,10, 70 9 of 19
features that are common to AGPs include the presence of branched type II arabino-
3,6-galactans (AGs) and short oligoarabinosides(both O-linked to the hydroxyproline
(Hyp) residues), a high percentage of amino acids that constitute the AG-II glycomodules
(Pro/Hyp, Ala, Ser, Thr, and Gly), N-terminal signal peptide directing their synthesis via
secretory pathway, and often a C-terminal glycosylphosphatidylinositol (GPI) lipid anchor
signal peptide [
93
,
94
]. While many AGPs are GPI-anchored to the plasma membranes,
others may be secreted to the medium [
93
,
95
]. Dragi´cevi´c et al. [
89
] recently demonstrated
that many AGP sequences may have transmembrane domains. Beside these basic struc-
tural features’ characteristic for classical AGPs and their short counterparts, AG peptides,
many AGPs contain additional conserved domains or functional motifs and are termed
chimeric AGPs [94].
AGPs are involved in cell proliferation [
96
] and diverse developmental and phys-
iological processes, including differentiation and patterning [
93
,
95
,
97
]. Involvement of
AGPs in SE has been described in many plant species, such as maize [
98
], chicory [
99
],
Trifolium nigrescens [
100
], and others. As discussed below, the role of AGPs during SE from
centaury roots [
101
,
102
] and leaf explants [
103
,
104
] has been investigated using a variety of
approaches. One of the main tools for studying the AGPs’ functions, used for decades, is a
synthetic red dye,
β
-D-glucosyl Yariv reagent or
β
GlcY [
105
]. Most AGPs specifically bind
β
GlcY [1,3,5-tris (4-
β
-D-glycopyranosyloxyphenylazo)-2,4,6-trihydroxybenzene]; for
β
-
galactosyl Yariv reagent, similar to
β
GlcY, a noncovalent interaction with
β
-1,3-galactan
moieties of AGPs has been demonstrated [106].
β
GlcY has been widely used as a histochemical reagent to detect AGPs int issue
sections [
107
]. When C. erythraea roots are used for SE induction on a hormone-free
medium, initially, the whole root explants were stained with
β
GlcY, but the most intense
staining was in the epidermal cells and vascular tissue [
101
,
102
]. After one week in
culture,
β
GlcY intensively stained AGPs in the surface cell layers of the centaury root
explants, where somatic embryos were likely to develop. A similar staining pattern was
observed in the outer epidermal cells during SE induction in chicory root culture [
99
].
Considering that somatic embryos originate directly from the root epidermal cells [
50
],
the accumulation of AGPs in this region is indicative of their involvement in SE initiation.
After two weeks in culture, the subepidermal layers of root explants also reacted with
β
GlcY,
but neither developing globular embryos nor adventitious buds (which form alongside the
embryos) showed significant precipitation of AGPs with
β
GlcY [
102
]. Finally, after 8 weeks
in root culture, the epidermal and subepidermal cells were deeply stained with
β
GlcY,
while staining of vascular tissue was less intense. This is shown in the root cross-section
(Figure 4a), where several developed somatic embryos, as well as adventitious buds,
can be seen.
Plants 2020, 9, x FOR PEER REVIEW 12 of 20
commonly unavailable. Thus, we initially used in-house assembled centaury leaf and
root transcriptomes [113] and mined centaury AGP sequences using a homology-based
search. Using this approach, we have identified four centaury AGP transcripts, named
CeAGP1 through CeAGP4 [103]. Of these, CeAGP1, CeAGP2, and CeAGP4(GenBank:
KC733882, KC733883, and KC733885, respectively) were characterized with conserved
fasciclin domains and represented members of a subclass of chimeric AGPs known as
fasciclin-like AGPs or FLAs [114]. CeAGP1 was highly induced (26.7-fold) during mor-
phogenesis from centaury leaf explants in the light, where ISE was accompanied with
indirect shoot development, but more importantly, it was over 20 fold induced during
ISE in darkness, in comparison to the control explants, indicating its importance during
ISE [103].CeAGP2 was slightly induced during both direct (on hormone-free medium)
and indirect morphogenetic paths (ISE and indirect shoot development on inductive
media), while the induction of CeAGP4 during ISE and organogenesis on inductive me-
dia was very low [103]. The role of CeAGP1 in ISE can be viewed in light of the general
role of FLAs as molecules involved in cell adhesion and protein–protein interactions
[114]. CeAGP3 is an AG peptide with a conserved DUF1070 domain (GenBank:
KC733884, protein:AGN92423). The expression pattern ofCeAGP3 indicated its general
involvement indifferent morphogenetic paths in centaury, since this transcript was in-
duced 36.6-fold relative to control during ISE in darkness, but was also highly induced
during indirect morphogenesis in the light (ISE and organogenesis), as well as in direct
organogenesis on a hormone-free medium [103]. We have analyzed all 271 sequences
containing the DUF1070 domain (DUF stands for Domain of Unknown Function) from
25 diverse families of vascular plants, aiming to elucidate the function of this motif. As it
turned out, most of the DUF1070 domain represented typical glycosylphosphatidylino-
sitol lipid anchor signal peptide (GPIsp) found in short AGPs (AG peptides), so the
DUF1070 was renamed to arabinogalactan peptide (PF06376) [115]. To our best
knowledge, DUF1070/PF06376 is the only conserved domain exclusively found in AGPs
and HRGPs, in general. GPI anchors in proteins, such as CeAGP3, are proposed to in-
crease lateral mobility of the anchored proteins in the plasma membrane, allow polar-
ized targeting to the cell surface, inclusion in lipid rafts, as well as further processing by
GPI-specific phospholipases and glycosidases, thereby releasing diffusible AGPs and/or
carbohydrates as extracellular signals, as well as biologically active lipids as intracellular
signals [91,94,95,97,103,115]; any of the proposed features for GPI-anchored AGPs may
be important for morphogenesis and SE.
Figure 4. Distribution of AGPs during SE in centaury. (a)Cross-section of a root explant with somatic embryos at its sur-
face, stained with βGlcY reagent. Scale bar indicates 80 µm (b) Indirect SE on centaury leaf explants grown on 100 µM
βGlcY reagent in darkness. Somatic embryos form only on the parts of the explants that are not in direct contact with the
medium. (c)Embryogenic globule developed on leaf explant and labeled with JIM15 antibody. Scale bar indicates 10 µm.
Figure 4.
Distribution of AGPs during SE in centaury. (
a
) Cross-section of a root explant with somatic embryos at its surface,
stained with
β
GlcY reagent. Scale bar indicates 80
µ
m (
b
) Indirect SE on centaury leaf explants grown on 100
µ
M
β
GlcY
reagent in darkness. Somatic embryos form only on the parts of the explants that are not in direct contact with the medium.
(c) Embryogenic globule developed on leaf explant and labeled with JIM15 antibody. Scale bar indicates 10 µm.
Plants 2021,10, 70 10 of 19
To study the role of AGPs during morphogenesis
in vitro
,
β
GlcY can be applied as an
adjuvant to the culture medium [
96
,
99
,
108
,
109
]. Inactivation of AGPs by
β
GlcY binding
during the induction of SE in different systems, commonly inhibits SE [99] and/or affects
embryos’ development and morphology [
108
,
109
]. As expected, the addition of
β
GlcY to
the inductive medium during the induction of ISE from centaury leaf explants in darkness
reduced the number of developed somatic embryos per explant in a dose-dependent man-
ner [
103
]. The concentration of 150
µ
M
β
GlcY almost completely inhibited ISE, whereas,
at lower concentrations, the embryos developed only in the explants’ regions that were
not in direct contact with the
β
GlcY-containing medium (Figure 4b). Indirect shoot de-
velopment on the same inductive medium in the light or direct shoot development on a
hormone-free medium were also inhibited by
β
GlcY in a concentration-dependent manner
but were less sensitive to
β
GlcY than ISE [
103
]. The obtained results clearly point at AGPs
as essential factors during ISE from centaury leaf explants.
However, quite unexpected results were obtained when
β
GlcY was used to investigate
the role of AGPs during morphogenesis (simultaneous development of somatic embryos
and adventitious buds) from centaury root explants [
101
,
102
]. Namely, it turned out that
β
GlcY may actually stimulate the morphogenesis from the root explants, albeit not in a
linear dose-response manner.
β
GlcY increased the shoot regeneration frequency of roots
cultured on the hormone-free medium from 71.67% for untreated culture to 93.89% and
92.22% for cultures grown on 15
µ
M or 25
µ
M
β
GlcY, respectively. The same concentrations
also increased the average number of regenerated shoots per root explant, while lower
(5
µ
M) or higher (50–75
µ
M)
β
GlcY concentrations had little effect on the regeneration po-
tential of the 8-week-old root culture in comparison to untreated control [
101
]. Comparable
results were obtained when the regeneration was scored after four weeks in culture [
102
]
or when 1
µ
M IBA was added to the medium [
100
]. In any case, the obtained regenerants
displayed normal morphology. Interestingly, the shoots regenerated on media containing
25–75
µ
M
β
GlcY had elevated content of AGPs in comparison to control shoots [
101
],
as determined by the single radial gel diffusion method, which also utilizes
β
GlcY [
110
].
The roots developed on regenerated shoots also had increased AGP levels when developed
on
β
GlcY-containing media [
101
]. This finding suggests that blocking of AGPs may increase
their synthesis by some type of feedback regulation. The authors suggested that
β
GlcY
in tissue culture may act as a stressor that may stimulate regeneration [
102
] since
β
GlcY
triggers wound-like responses in Arabidopsis cell culture, as shown by whole-genome
array [
111
]. On the other hand, the presence of 75
µ
M
β
GlcY in the centaury 4-weeks old
leaf culture did not alter the AGP content, regardless of other conditions (basal or inductive
medium and light vs. darkness) [
103
], so the effect of
β
GlcY on AGPs accumulation might
be tissue-specific. Finally, the profile of AGPs present in the regenerating leaf explants,
as determined by crossed electrophoresis [
112
], depends on the medium composition,
light conditions, and culture age [103].
7. Dynamic Changes of AGPs Distribution and Expression during SE in Centaury
Overall, the evidence collected using
β
GlcY reagent in different assays suggests that
AGPs are important for the induction of SE in centaury. However, tracking the dynamicsof
AGPs’ distribution during regeneration requires more sophisticated methods, such asa
widely use dimmunohistochemical approach with monoclonal antibodies (mAbs) raised
against AGPs’ carbohydrate epitopes.A large set of the anti-AGP mAbs of the JIM, LM,
and MAC series are commercially available and are listed, along with the epitopes they
recognize, in several reviews [
95
,
96
]. The exact structure of the recognized epitopes is not
always clearly determined, but it is known that MAC207, JIM4, and JIM13 bind to the
β
-D-
GlcA-(1
→
3)-
α
-D-GalA-(1
→
2)-
α
-L-Rha motif, whereasLM2 recognizes
β
-linked glucuronic
acid (
β
-D-GlcA). A systematic review of the available literature describing the expression
of different mAb-recognizable AGP epitopes during SE in different species [
98
–
100
,
109
],
to mention a few, would surely transcend the scope of the current review and would only
confirm that none of the tested epitopes stands out as a universal SE marker in all or most
Plants 2021,10, 70 11 of 19
of the studied plant species. Thus, the existence of two systems for SE induction in the
same species—DSE on the hormone-free medium from root explants [
102
] and ISE from
the leaf explants cultured on inductive medium [
104
]—provides an opportunity for the
comparison of the obtained immunohistochemical results and search for common epitope
markers or patterns.
The morphogenesis from the root explants was studied using LM2, JIM13, JIM15,
JIM16, and MAC207 mAbs, of which the expression of JIM13-reactive epitope was not
detected at all [
102
]. The JIM16 epitopes were localized in all cells of the root explants,
especially in the endodermis and the central cylinder, as well as in the newly formed
meristematic centers, so they were considered as markers of organogenesis, not somatic
embryogenesis. The remaining three mAbs reacted with the epitopes present in somatic
embryos. LM2 epitopes were widely distributed in root cross-sections at the beginning of
the culture, but after 4 weeks in culture, the LM2 signal was more localized in epidermal
cells and newly formed globular somatic embryos [
102
]. Comparable results for LM2
localization were obtained during DSE from chicory roots [
99
], where this epitope was
foundin the surface cell layer surrounding somatic embryos; however, in this system,
JIM13 and JIM16 mAbs were also expressed. MAC207 epitope had strong expression
in protodermal cells of the embryos, as well as at the surface of epidermal cells of root
explants adjacent to globular somatic embryos, with a strong signal in the extracellular
matrix. Finally, the JIM15 epitope was reactive with AGPs in developed somatic embryos,
as well as in the cells of the vascular elements of the root explants [102].
A slightly different set of mAbs, comprisingJIM4, JIM8, JIM13, JIM15, LM2, LM14,
and MAC207, was used to investigate the distribution of the corresponding AGP epi-
topes during ISE from C. erythraea leaf explants [
104
]. As discussed above, in this system,
light induces simultaneous development of both somatic embryos and adventitious buds,
whereas in darkness, only ISE occurs. Generally, in globular somatic embryos, a different
distribution pattern of JIM4, JIM13, JIM15, LM2, and MAC207 epitopes was observed,
while with the progression of SE, the number of detected AGPs decreased. When the
explants are cultivated in darkness, the JIM4 epitope was strongly expressed from the
earliest stages of SE: It localized in the epidermal and subepidermal cells which formed
meristematic centers, in the embryogenic cells in meristematic calli, as well as in four-cell
proembryo. During further proembryo development, strong expression was detected only
in the extracellular matrix surrounding the proembryogenic nodule. At the globular stage,
JIM4 epitopes were found in the cell walls of the protodermal cells, while at the early
cotyledonary stage, the JIM4 fluorescence was moderate [
104
]. Even though this mAb
was not exclusively present in embryogenic tissues, its expression in adventitious buds
formed in the light was weak. In maize callus culture, JIM4 was as also an early marker
of embryogenic competence [
98
]. A similar labeling pattern to JIM4 in globular somatic
embryos was found for MAC207 since its strong signal was detected in the cell walls of
protodermal cells [
104
], just as was seen in protodermal cells of the globular embryos
developed from roots [
102
].The JIM13 epitope, which was not observed during SE from
the root explants at all [
102
], showed an intense signal in the whole globular embryos
developed from leaf explants in darkness but was not restricted only to the embryogenic
tissues [
104
]. The expression of JIM13 decreased in late embryos. In adventitious buds
developed in the light, this epitope was not detected. Strong JIM13 labeling was also found
in the embryogenic sector during SE in peach palm, where it was associated with extracel-
lular matrix surface network [
108
]. Likewise, high-intensity JIM15 (Figure 4c) and LM2
fluorescence were localized to the whole globular embryos, but not in later developmental
stages. A strong LM2 signal was observed in the cell walls of meristematic cells from which
somatic embryos develop and in cells of embryogenic swellings in Trifolium nigrescens [
100
].
Both JIM15 and LM2 signals were also seen in developing adventitious buds, so they
were not an exclusive feature of SE. Unlike other tested epitopes, which appeared early
during ISE, the LM14 signal was not present in globular somatic embryos but was strong
and evenly distributed throughout the longitudinal sections of the heart embryo. Finally,
Plants 2021,10, 70 12 of 19
theJIM8 epitope was detected in the extracellular matrix, as well as in adventitious buds,
but not in somatic embryos of any stage [104].
The obtained immunohistochemical results only corroborated well-established obser-
vations that spatiotemporal occurrence of AGPs during SE is developmentally regulated
and that AGPs may serve as positional markers, markers of cell identity, or markers for
embryogenic competence [
97
–
100
]. Our results indicate that the profile of AGP epitopes
expressed during SE is not only species-specific but also strongly depends on the explant
type and the culture conditions: While some epitopes, such as MAC207, have similar
expression patterns in both regeneration systems, others, such asJIM13, are strongly ex-
pressed in somatic embryos developed from leaves, but are absent in embryos regenerated
on C. erythraea roots. Furthermore, even though some mAbs recognize the same epitope
(MAC207, JIM4, and JIM13), they display different labeling patterns.
Even though anti-AGP mAbs have been widely used for studying AGPs’ distribution
during SE and other developmental processes, their usefulness is intrinsically limited for
several reasons: (1) mAbs are not specific for a single AGP; (2) they cannot distinguish all
glycoforms of an AGP backbone, and (3) the epitope has to be unmasked for immunodetec-
tion [
89
,
91
,
92
]. Of course, that the analysis of the spatiotemporal pattern of gene expression
can indicate the involvement of a particular AGP in some process [
93
], but in non-model
species, such as C. erythraea, the necessary sequence resources are commonly unavailable.
Thus, we initially used in-house assembled centaury leaf and root transcriptomes [
113
]
and mined centaury AGP sequences using a homology-based search. Using this approach,
we have identified four centaury AGP transcripts, named CeAGP1 through CeAGP4 [
103
].
Of these, CeAGP1,CeAGP2, and CeAGP4 (GenBank: KC733882, KC733883, and KC733885,
respectively) were characterized with conserved fasciclin domains and represented mem-
bers of a subclass of chimeric AGPs known as fasciclin-like AGPs or FLAs [
114
]. CeAGP1
was highly induced (26.7-fold) during morphogenesis from centaury leaf explants in the
light, where ISE was accompanied with indirect shoot development, but more importantly,
it was over 20 fold induced during ISE in darkness, in comparison to the control explants,
indicating its importance during ISE [
103
]. CeAGP2 was slightly induced during both
direct (on hormone-free medium) and indirect morphogenetic paths (ISE and indirect shoot
development on inductive media), while the induction of CeAGP4 during ISE and organo-
genesis on inductive media was very low [
103
]. The role of CeAGP1 in ISE can be viewed in
light of the general role of FLAs as molecules involved in cell adhesion and protein–protein
interactions [
114
]. CeAGP3 is an AG peptide with a conserved DUF1070 domain (GenBank:
KC733884, protein:AGN92423). The expression pattern of CeAGP3 indicated its general
involvement indifferent morphogenetic paths in centaury, since this transcript was induced
36.6-fold relative to control during ISE in darkness, but was also highly induced during
indirect morphogenesis in the light (ISE and organogenesis), as well as in direct organogen-
esis on a hormone-free medium [
103
]. We have analyzed all 271 sequences containing the
DUF1070 domain (DUF stands for Domain of Unknown Function) from 25 diverse families
of vascular plants, aiming to elucidate the function of this motif. As it turned out, most of
the DUF1070 domain represented typical glycosylphosphatidylinositol lipid anchor signal
peptide (GPIsp) found in short AGPs (AG peptides), so the DUF1070 was renamed to
arabinogalactan peptide (PF06376) [
115
]. To our best knowledge, DUF1070/PF06376 is the
only conserved domain exclusively found in AGPs and HRGPs, in general. GPI anchors
in proteins, such as CeAGP3, are proposed to increase lateral mobility of the anchored
proteins in the plasma membrane, allow polarized targeting to the cell surface, inclusion in
lipid rafts, as well as further processing by GPI-specific phospholipases and glycosidases,
thereby releasing diffusible AGPs and/or carbohydrates as extracellular signals, as well as
biologically active lipids as intracellular signals [
91
,
94
,
95
,
97
,
103
,
115
]; any of the proposed
features for GPI-anchored AGPs may be important for morphogenesis and SE.
Plants 2021,10, 70 13 of 19
8. Perspectives: Novel SE Markers, “AGP-Tyr Kinases”, and Time-Laps Embryogenesis
To support the analysis of the molecular events during SE and other
in vitro
morpho-
genetic processes in centaury, we have recently sequenced six C. erythraea transcriptomes
(embryogenic calli, globular somatic embryos, cotyledonary somatic embryos, adventi-
tious buds, leaves and roots of
in vitro
grown plants) and de novo assembled referent
transcriptome comprising 105.726 genes [
116
]. The high quality and completeness tran-
scriptome were functionally annotated and made publicly available. The transcriptome,
along with a set of validated housekeeping genes, comprises a framework for the search for
genes involved in SE and organogenesis [
116
]. A subset of genes potentially involved is SE
was selected as transcripts with
≥
8-fold higher expression (FPKM values) in embryogenic
tissues as compared to non-embryogenic tissues, and their expression was further analyzed
by qRT-PCR in 16 tissue samples [
117
]. The most intriguing finding of this preliminary
research was the expression profile an unknown gene (provisionally termed UN1), a 725 bp
long transcript with no BLAST hits or homology with any known sequence. UN1 was
highly expressed in leaf-derived embryogenic calli, while its expression progressively
decreased in globular and cotyledonary embryos. The UN1 expression in seedlings, roots,
leaf-derived adventitious buds and leaves from flowering plants was below the qPCR
detection limits, implying that its expression is restricted to the initial ISE stages [
11
].
The investigation of UN1 structure and function is ongoing; for now, we can only speculate
that UN1 may have an impact on the acquisition of the embryogenic potential, and as such
it may be a novel SE marker.
As discussed above, a homology-based search revealed only four AGP sequences in
the first version of the C. erythraea transcriptome, all of which were AGPs with conserved
domains [
103
]. This was expected since HRGPs, including AGPs, are intrinsically disor-
dered proteins lacking hydrophobic core, so the sequence constraints imposed on these
proteins are relatively low. Therefore, AGPs can rapidly mutate and evolve, which hinders
their homology-based mining [
90
]. We have recently developed a highly sophisticated
bioinformatics pipeline developed in R, ragp, for mining and analysis of HRGPs with an
emphasis on AGPs [
90
]. The key novelty incorporated in ragp is the machine learning-
based prediction of proline hydroxylation sites, which represent the glycosylation sites.
The analysis of C. erythraea transcriptome [
118
] as well as 62 plant proteomes using ragp [
90
]
revealed, quite unexpectedly, that the most frequently identified domains found in AGPs
were the Protein kinase and Protein tyrosine kinase domains. The Protein (tyrosine) kinase
domains have thus far eluded experimental evidence for linkage with AGPs in any plant
species. Possible implications of this finding include a novel way of attachment of AGPs
to the plasm membrane through their transmembrane domains and a novel way for the
involvement of AGPs in signaling. So far, structural features of AGPs and circumstantial
evidence suggested that AGPs may be involved in signaling as co-receptors [
97
], or through
interaction with membrane receptors (including protein kinases) on the same or neighbor-
ing cell [
11
,
91
,
93
], interaction with other AGPs, pectins, and other cell wall or cytoskeletal
elements [
89
,
93
]. The presence of protein kinase domains on ragp-predicted AGPs suggests
that AGPs may actually be the membrane receptors themselves or that certain Protein
kinases have previously undetected AG-glycomodules and can be glycosylated. While the
experimental evidence for the Hyp-glycosylation of these protein kinases is lacking, and the
functions of the proposed “AGP-Protein kinase” molecules are unknown, it should be
noted that, for example, A. thaliana SERK5 (AT2G13800.1) has predicted hydroxyprolines
organized in characteristic AG-glycomodules [
90
,
118
] (
Figure 5
). The analysis of expression
and function of “AGP-Protein kinases” and their possible involvement in SE in C. erythraea
is planned.
Since both DSE from centaury roots [
35
,
50
] and ISE from leaf explants [
28
] are asyn-
chronous, collecting embryogenic tissues, specifically somatic embryos at different develop-
mental stages for molecular and biochemical analyses, is very tedious and time-consuming.
Unfortunately, the establishment of a synchronized embryogenic culture has not been
achieved in C. erythraea yet, and it remains one of our goals. A synchronized culture would
Plants 2021,10, 70 14 of 19
not only aid the harvest of somatic embryos at a specific stage but would also indicate the
exact timing to a specific developmental event under certain conditions. An alternative
way to achieve this is documentation of the development of embryogenic structures on
selected explants over time. Such documentation system has been established by the com-
bination of photography (using a smartphone camera with a macro lens), image processing
of focalstacks from the developing explants automated in Adobe Photoshop and Bridge,
and a relational database built using Excel and R [
119
]. An example of such a time-lapse
documentation video of SE from leaf explants is provided as a Supplement.
Plants 2020, 9, x FOR PEER REVIEW 14 of 20
Figure 5. Arabinogalactan proteins (AGPs) with Tyr kinase domains. The first sequence is Somatic Embryogenesis Re-
ceptor-like Kinase 5 (SERK5) from A. thaliana (AT2G13800.1 or Q8LPS5 protein precursor). Four sequences below are
found in the C. erythraea transcriptome, based on homology with SERK5. In addition to AG glycomodules with predict-
ed hydroxyprolines and Tyr kinase domains, all sequences have N-terminal signal peptide and a transmembrane do-
main, while most have Leucine-rich repeats typical for SERK receptors. The image is generated using ragp.
Since both DSE from centaury roots [35,50] and ISE from leaf explants [28] are
asynchronous, collecting embryogenic tissues, specifically somatic embryos at different
developmental stages for molecular and biochemical analyses, is very tedious and
time-consuming. Unfortunately, the establishment of a synchronized embryogenic cul-
ture has not been achieved in C. erythraea yet, and it remains one of our goals. A syn-
chronized culture would not only aid the harvest of somatic embryos at a specific stage
but would also indicate the exact timing to a specific developmental event under certain
conditions. An alternative way to achieve this is documentation of the development of
embryogenic structures on selected explants over time. Such documentation system has
been established by the combination of photography (using a smartphone camera with a
macro lens), image processing of focalstacks from the developing explants automated in
Adobe Photoshop and Bridge, and a relational database built using Excel and R [119].
An example of such a time-lapse documentation video of SE from leaf explants is pro-
vided as a Supplement.
9. Conclusions
Even after 20 years of research, Centaurium erythraea remains an attractive, chal-
lenging, and yet rewarding experimental object at our Department. C. erythraea is already
firmly established as a valued model system at our Department for the studies on alter-
native ways for the production of secondary metabolites and for the studies on mor-
phogenesis in vitro—both primarily aimed at its conservation and sustainable usage.
However, the accumulated data and successful protocols for the centaury propagation in
vitro [28] have led us to gradually shift our focus from centaury’s potential as a medicinal
plant to its possibly even greater potential as a genetic resource for crop improvement.
Namely, we believe that centaury’s immense regeneration potential and developmental
plasticity, when cultivated in vitro, rely on the presence or high activity of certain genes
that may not be present or active in plant species recalcitrant to SE induction or in vitro
propagation and manipulations in general. Having a sequenced C. erythraea transcrip-
tome [116] would allow us, and other research groups interested in centaury develop-
ment, to mine for genes that are highly active during SE and organogenesis, and hope-
fully find genes, such as UN1 [117], that were not described before. Such novel genes, as
well as known genes previously unassociated with morphogenesis, could be considered
as sequence resources for the genetic improvement of valuable crops that are recalcitrant
to in vitro manipulations. In addition, finding genes that are differentially expressed
during DSE and ISE from roots and leaves, respectively, specifically AGP genes and
genes associated with auxin and cytokinin signaling or metabolism, as well as differences
Figure 5.
Arabinogalactan proteins (AGPs) with Tyr kinase domains. The first sequence is Somatic Embryogenesis
Receptor-like Kinase 5 (SERK5) from A. thaliana (AT2G13800.1 or Q8LPS5 protein precursor). Four sequences below are
found in the C. erythraea transcriptome, based on homology with SERK5. In addition to AG glycomodules with predicted
hydroxyprolines and Tyr kinase domains, all sequences have N-terminal signal peptide and a transmembrane domain,
while most have Leucine-rich repeats typical for SERK receptors. The image is generated using ragp.
9. Conclusions
Even after 20 years of research, Centaurium erythraea remains an attractive, challenging,
and yet rewarding experimental object at our Department. C. erythraea is already firmly
established as a valued model system at our Department for the studies on alternative
ways for the production of secondary metabolites and for the studies on morphogene-
sis
in vitro
—both primarily aimed at its conservation and sustainable usage. However,
the accumulated data and successful protocols for the centaury propagation
in vitro
[
28
]
have led us to gradually shift our focus from centaury’s potential as a medicinal plant to
its possibly even greater potential as a genetic resource for crop improvement. Namely,
we believe that centaury’s immense regeneration potential and developmental plasticity,
when cultivated
in vitro
, rely on the presence or high activity of certain genes that may not
be present or active in plant species recalcitrant to SE induction or
in vitro
propagation
and manipulations in general. Having a sequenced C. erythraea transcriptome [
116
] would
allow us, and other research groups interested in centaury development, to mine for genes
that are highly active during SE and organogenesis, and hopefully find genes, such as
UN1 [
117
], that were not described before. Such novel genes, as well as known genes
previously unassociated with morphogenesis, could be considered as sequence resources
for the genetic improvement of valuable crops that are recalcitrant to
in vitro
manipula-
tions. In addition, finding genes that are differentially expressed during DSE and ISE from
roots and leaves, respectively, specifically AGP genes and genes associated with auxin and
cytokinin signaling or metabolism, as well as differences in the endogenous hormones
during these two processes, would probably highlight some factors governing SE via direct
or indirect pathway. Thus, unrevealing at least a part of the molecular networks and genes
Plants 2021,10, 70 15 of 19
that are at the base of SE induction and other regeneration processes in centaury is the
primary focus for our future research.
Supplementary Materials:
The following are available online at https://www.mdpi.com/2223-774
7/10/1/70/s1, Time laps photo-documentation of somatic embryogenesis from Centaurium erythraea
leaf culture is provided in a form of a video file Centaury somatic embryogenesis.mp4. The size of
the video is about 25 Mb.
Author Contributions:
Conceptualization, A.R.S., A.D.S., M.M.T.-M., and B.K.F.; writing—original
draft preparation, Sections 1and 2, A.R.S.; writing—original draft preparation,
Sections 3and 4,
M.M.T.-M. and M.P.M.; writing—original draft preparation, Section 5, B.K.F.; writing—original
draft preparation, Sections 6and 7, A.D.S.; writing—original draft preparation, Section 8, A.D.S.
and M.D.B.; writing—review and editing, A.D.S.; visualization, all authors.; Supplementary video,
M.D.B.; supervision, A.R.S. All authors have read and agreed to the published version of the
manuscript.
Funding: This research received no external funding.
Acknowledgments:
This work was supported by the Ministry of Education, Science and Technological
Development of the Republic of Serbia, contract number 451-03-68/2020-14/200007. The authors are
grateful to Milan Dragi´cevi´cfor Figure 5generated in ragp. The authors also thank Milica Simonovi´c
for rendering and post-production of the somatic embryogenesis time-laps video (Supplement).
Conflicts of Interest: The authors declare no conflict of interest.
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