Mol Biol Rep (2010) 37:24932507
DOI 10.1007/s11033-009-9764-3
The molecular basis for stress-induced acquisition of somatic
embryogenesis
Omid Karami Abbas Saidi
Received: 8 October 2008 / Accepted: 14 August 2009 / Published online: 25 August 2009
Springer Science+Business Media B.V. 2009
Abstract Somatic embryogenesis (SE) has been studied
as a model system for understanding of molecular events in
the physiology, biochemistry, and biology areas occurring
during plant embryo development. Stresses are also the
factors that have been increasingly recognized as having
important role in the induction of SE. Plant growth regu-
lators such as 2,4-dichlorophenoxyacetic acid (2,4-D),
ABA, ethylene, and high concentrations of 2,4-D are
known as stress-related substances for acquisition of
embryogenic competence by plant cells. Gene expression
analysis in both the proteome and transcriptome levels
have led to the identification and characterization of some
stress-related genes and proteins associated with SE. This
review focuses on the molecular basis for stress-induced
acquisition of SE.
Keywords Stress
Somatic embryogenesis
Gene

Protein
Introduction
Embryogenesis is a crucial developmental process in the
life cycle of plants spanning the transition from the fertil-
ized egg to the generation of a mature embryo. In this
process the embryo acquires a defined apical-basal pattern
along the main body axis with shoot and root poles, a
O. Karami (&)
Department of Biotechnology, Bu-Ali Sina University,
Hamadan, Iran
e-mail: hiva@basu.ac.ir
A. Saidi
Department of Biotechnology, Shahid Beheshti University,
Tehran, G.C., Iran
hypocotyl and cotyledons. Somatic embryogenesis (SE) is
the developmental restructuring of somatic cells toward the
embryogenic pathway and forms the basis of cellular toti-
potency in higher plants [72, 128]. It is the capacity of
somatic cells to form, in culture, complete new embryos
via a process that resembles zygotic embryogenesis. The
developmental process of SE shares considerable similarity
with that of zygotic embryogenesis [18, 196], probably due
to conservation in the underpinning cellular and molecular
mechanisms between the two processes. SE provides an
attractive model system for studying zygotic embryogen-
esis, particularly because zygotic embryos are encased by
maternal tissues and are difficult to access using bio-
chemical and molecular tools. In contrast to zygotic
embryogenesis, SE is a nonsexual propagation process
where somatic cells differentiate somatic embryos. There-
fore, somatic embryos can be used for studying the regu-
lation of embryo development. One advantage with
somatic embryos is that the developmental process can be
controlled and synchronized so that sufficient quantities of
developmentally homogeneous tissue can be collected at
specific stages. However, SE has been viewed as a tool for
massive propagation of commercial crops and as a poten-
tial model system for the study of the regulation of gene
expression required for the earliest developmental events in
the life of higher plants, such as the developmental
mechanism of embryogenesis [93, 196]. In addition, as the
initial basis of cellular and genetic engineering, SE plays
an important role in genetic transformation, somatic
hybridization, and somaclonal variation.
The induction of SE in cultured tissue is a multi-facto-
rial event. Induction by plant hormones, particularly auxin,
is generally considered to be the most important factor
[147] and is by far the most widely studied. Other chemical
factors, such as nutritional components of culture medium
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2494 Mol Biol Rep (2010) 37:24932507

and physical factors such as lighting or the form of culture
medium liquid or solid, have also been shown to play a role
in some species. Another factor, which has become
increasingly recognized in recent years, is stress (Fig. 1). It
is clear that excising tissue from the parent plant, sub-
jecting to sterilization procedures, cutting into pieces and
putting into an artificial culture environment exerts con-
siderable stress on the plant tissue. The connection between
stress and SE has received increasing attention over recent
years. There are some evidence that the stressresponse of
cultured tissue plays a major role in somatic embryo
induction [42, 60, 78, 79, 90, 102, 128, 175]. However,
embryogenic competence of in vitro cultured somatic cells
can be stimulated by various factors, such as heavy metal
ions [73, 136], high osmotic pressure [1, 73, 78, 82, 83, 90,
193], dehydration [98, 136], explant wounding [17, 151]
and high temperature [79, 93].
Plant SE is a complicated developmental process
involving the acquisition of embryogenic ability by
somatic cells, as well as their dedifferentiation and repro-
gramming gene expression patterns that engages cascades
of genetic triggers turning on and off the expression of
specific genes [42, 122]. One of the hypothesis on mech-
anisms involved in stress-induced embryogenesis
highlights the importance of the interaction between auxin
and stress signaling which results in acquiring the
embryogenic competence of somatic cell by broad cellular
reprogramming manifested at different levels [42]. Early
phases of SE are characterized by the induction of many
stress-related genes, which leads to the hypothesis that SE
is an extreme stress response of cultured plant cells [33].
However, the mechanism by which these stress treatments
stimulate dedifferentiation into embryogenically competent
cells is still unknown. In light of the increasing evidence of
a role of stress in the induction of SE, it is necessary to take
into account the stress-response of cultured tissue when
investigating the genetic regulation of SE.
No review has so far been published with the role of
stress in SE. In this review, information on stress-related
molecular basis associated with induction of SE are
described.
Role of 2,4-D
Auxin is considered to be a positional and pattering signal
molecule that plays a major role in zygotic embryogenesis
[165] as well as SE [19, 42, 134]. The concentrations of

Fig. 1 Stress induction of

somatic embryos in carrot,
Arabidopsis, and carnation. a
Induction of somatic embryo on
apical tip segments of carrot
seedlings were by stress
conditions (0.7 M sucrose,
0.3 M NaCl or a high
temperature of 37C). b
Somatic embryos formed from
apical tip segments of seedlings
of Arabidopsis by 0.7 M
sorbitol. c SE induction in
carnation from immature petals
by 0.6 M mannitol

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Mol Biol Rep (2010) 37:24932507
exogenously applied auxin, required to induce SE, are
much higher than the endogenous auxin levels in plant
tissues [60, 77, 146]. Among different auxins, 2,4-D was
the most commonly used for the induction of somatic
embryogenesis. In more than 65% of the recent protocols,
2,4-D alone or in combination with other plant growth
regulators and also in many in vitro SE systems has been
employed as an inducer [48]. Many reports have been
shown that the formation of an embryogenic cell is related
to nuclear DNA hypermethylation in the presence of 2,4-D
[103, 105, 191]. In Cucurbita pepo the highest rate of DNA
methylation occurred in the early embryo stages, predom-
inantly on medium containing 2,4-D, and DNA methyla-
tion decreased during embryo maturation on auxin-free
medium [103]. A carrot DNA methyltransferase gene,
MET1-5 was expressed transiently after the induction of SE
by 2,4-D, before the formation of embryogenic cell clumps,
and 5-azacytidine, an inhibitor of DNA methylation, sup-
pressed the formation of embryogenic cell clumps from
epidermal carrot cells [190]. Arabidopsis plants with an
antisense MET1 transgene, partial-loss-of-function met1
mutations, or chromomethylase3, domains rearranged
methylase1 and domains rearranged methylase1 mutations
revealed that reduced DNA methylation results in abnor-
mal postembryonic plant development [13, 44, 80, 84, 145,
188]. The question is how does DNA methylation affect
acquiring the embryogenic competence at the presence of
2,4-D? DNA methylation is a unique and noteworthy
process because it involves the covalent modification of a
cells genetic material [51, 94] and plays an important role
modifying the information content of the underlying
genetic sequence and gene expression [6]. Xiao et al. [188]
show that DNA methylation performed by methyltrans-
ferase1 influences gene expression during embryogenesis
in Arabidopsis. Therefore, dynamic changes in chromatin
structure by DNA methylation at presence of 2,4-D leads to
genomic reprogramming in somatic cells and hundreds of
genes specifically required for acquiring the embryogenic
competence are expressed (Fig. 2).
Fig. 2 A model showing
embryogenic competence by
DNA methylation at presence of
2,4-D. DNA methylation is
followed by chromatin
remolding in somatic cells.
Finally, somatic cells undergo
genomic reprogramming and
acquire the embryogenic
competence
2495
2,4-D may have several roles in this process, acting as
an auxin directly or modifying intracellular indole acetic
acid (IAA) metabolism and/or as a stressor [42, 143]. In
addition, 2,4-D is more effective at inducing SE than the
endogenous auxin IAA, because the artificial auxin cannot
be metabolized in plant cells. The state of rapid cell pro-
liferation and active aerobic metabolism occurring at the
presence of 2,4-D often leads to an oxidative burst in the
tissue through generating reactive oxygen species, includ-
ing hydrogen peroxide [138, 166]. These facts suggest that
when exogenous auxin is applied, it acts as a stressor rather
than a plant hormone. Many genes which are induced by
auxin also found to be responsive to various abiotic stres-
ses. For example, these may include genes encoding a
putative oxysterol binding protein, spermidine synthase,
salt inducible kinase, a-tubulin, step II splicing factor and
malate dehydrogenase. Triticum aestivum salt inducible
protein kinase (TaSIPK) transcripts were abundant both in
somatic embryos and zygotic tissues. The likely role of
TaSIPK in stress-induced signaling relates this pathway to
the auxin induced cascade leading eventually to the
induction of SE in T. aestivum. As of now, there is a sol-
itary report in Medicago, indicating its involvement in SE
[128]. 2,4-D is known to induce many stress-related genes
[24, 128, 132, 134, 142]. In soybean and potato, somatic
embryos are induced in cotyledons by 2,4-D and are
associated with up-regulation of oxidative stress and
defense genes [158, 172]. However, 2,4-D is also a strong
herbicide and the concentration of IAA required for the
induction of SE is over 103 times the endogenous free IAA
level [146]. Therefore, 2,4 D is thought to function as a
stress substance rather than a phytohormone, triggering the
acquisition of embryogenic competence by plant cells.
Role of ABA
The plant hormone abscisic acid (ABA) regulates many
important plant developmental processes, and induces
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epigenetic reprogramming against tolerance to different
stresses including drought, salinity, low temperature, and
some pathogens [54, 174]. ABA serves as a critical
chemical messenger for stress responses. Under abiotic
stress conditions, especially under drought and high
saline conditions, ABA biosynthesis is up-regulated [189].
Several studies have shown that ABA accumulation is
required for the development of stress tolerance in plants.
As a rapid response, stomatal closure is induced by ABA
accumulation.
There are several reports that ABA applications stimu-
lated SE [43, 104, 125, 129]. It was also reported that
embryonic cell clusters contained higher levels of endog-
enous ABA than non-embryonic calli [91, 120]. Tranthi
and Pleschka [176] reported a positive relationship
between the endogenous contents of ABA in petioles and
somatic embryogenesis of some Daucus species. A rela-
tionship between endogenous ABA and the induction of SE
was demonstrated using stress-induced system of somatic
embryos in carrot [86]. Endogenous levels of ABA also
increase in response to stress treatments in various plants
[17] including maize [149], pea [41], oilseed rape [153]
and carrot [76]. During androgenesis induction in barley by
a mannitol stress treatment higher regeneration efficiencies
have been correlated to increasing levels of osmotic stress
and ABA [70]. Kikuchi et al. [86] reported that somatic
embryo formation was inhibited by the application of
fluridone, a potent inhibitor of ABA biosynthesis, during
stress treatment. Since ABA is known to increase after
various stresses, it has been proposed that increased levels
of ABA might induce SE [152].
How does ABA affect in SE? Under abiotic stress con-
ditions, ABA biosynthesis is upregulated. Several b-ZIP
transcription factors in Arabidopsis are the direct targets of
protein phosphorylation by sucrose non-fermenting protein
(SNF1)-related kinase 2 (SnRK2), among which three are
activated by ABA [46]. These b-ZIP transcription factors
bind to the consensus motif ABA-responsive element
(ABRE) characterized by the core motif ACGTGGC found
in many promoters that are responsive to ABA [81]. b-ZIP
form the basic transcriptional platform to enlist other reg-
ulatory proteins on target promoters containing the ABRE
motif (Fig. 3). ABA-responsive gene expression in vege-
tative tissues has been shown to require calcium [160].
Elevation of calcium is sufficient to trigger ABA-responsive
gene expression [160]. Many studies implicate Ca2? as a
messenger in abiotic stress [148, 150]. Calcium is a central
regulator of plant cell signaling [66] and which has been
shown to play an important second messenger involved in
ABA signal transduction [40, 68]. Plants have several
classes of calcium sensory proteins, including calmodulin
and CaM-related proteins [107], calcineurin B-like proteins
[107], and calcium-dependent protein kinases (CDPKs)
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Mol Biol Rep (2010) 37:24932507
Fig. 3 A model depicting a possible ABA signaling pathway that
leads to transcriptional activation of a gene network. SnRK2 can
interact physically and phosphorylate b-ZIP transcriptional activators
using peptides or protein fragments containing motifs that are found
in the C domains. Also, b-ZIP transcriptional activators may be
phosphorylated by certain CDPKs, which then can recognize similar
or even identical C-domain motifs as the SnRK2s. The downstream
target genes are statistically enriched in ABRE sequences in their
promoters, which respond to ABA in gene reporter assays and are the
binding motifs of b-ZIP transcription factors
[16, 61]. CDPKs are encoded by large multigene family
with possible redundancy and/or diversity in their functions
[16, 61], and they are believed to be important components
in plant hormone signaling [16, 108]. Two CDPKs were
identified in soluble protein extracts of embryogenic cul-
tures of sandalwood [167]. The proteins showed differential
expression and were absent in plantlets regenerated from
somatic embryos. Expression of the MsCDPK3-encoding
gene has been shown to increase during the early phase
of embryogenesis from cultured alfalfa cells (23). Two
Arabidopsis CDPKs, AtCPK10, and AtCPK30 have been
demonstrated to activate a stress and ABA-inducible
promoter, showing the suggesting connection of CDPKs
to ABA signaling pathways [160]. b-ZIP transcriptional
activators may be phosphorylated by CDPKs, which rec-
ognize similar or even identical C-domain motifs as the
SnRK2s (Fig. 3). However, molecular genetic evidence via
gene disruption is needed to unequivocally link defined
CDPK genes with ABA-regulated biological functions
in SE.
Induction of carrot SE, by treatment with various
stresses, has been exploited to isolate those proteins and
genes that are thought to be related to the acquisition of
embryogenic competence. These proteins, i.e. embryogenic
cell proteins (ECPs), belong to the late embryogenesis-
abundant protein groups [92, 169]. Expression of the ECP
genes is positively regulated by ABA. The gene ABI3 was
isolated, based on the studies of ABA-insensitive Arabid-
opsis mutants the ABI3 gene was isolated [96]. This gene is
believed to be related to the seed-specific signal
Mol Biol Rep (2010) 37:24932507
transduction of ABA [131]. A homolog of this gene in
carrot was isolated and named C-ABI3 [162]. This gene is
mainly expressed in embryonic tissue and positively reg-
ulates the expression of the ECP genes [161, 162]. Kikuchi
et al. [86] reported that, C-ABI3 and ECPs were expressed
during stress treatment prior to the formation of somatic
embryos in carrot. These results suggest that the stress
induced accumulation of endogenous ABA is involved in
the induction of carrot SE.
However, in contrast to the case of auxin, we do not
yet have clear genetic and biochemical evidence for the
involvement of ABA in the induction and development
of plant embryogenesis. Studies on ABA physiological
responses are needed to clarify the molecular mecha-
nisms that trigger the acquisition of embryogenic
competence.
Role of ethylene
Synthesis of the growth regulator ethylene can be rapidly
evoked in response to a variety of biotic and abiotic stresses
including wounding [183]. Reports on the effects of ethyl-
ene on SE are variable and this is not surprising because
ethylene concentration and signaling interactions with other
hormones [135] are likely to be species developmentally
and environmentally dependent. 1-aminocyclopropane-
1-carboxylate (ACC) synthase was up-regulated on an
auxin-rich callus induction medium [15] in Arabidopsis.
However, in a defined experimental system of a develop-
mental process, as with other hormones, there are most
likely specific roles to play in the genetic networks [123].
Up-regulation of transcripts of ethylene biosynthesis
genes has also been seen in wounding [17, 27] and SE in
soybean cotyledons [172]. The first question that arises is
whether the ethylene biosynthesis genes really reflect a
requirement for ethylene for SE. Results with an inhibitor
of ethylene biosynthesis (AVG) and ethylene perception
(Ag?) strongly supported the idea that ethylene is essential
for SE in M. truncatula. Consistent with this, the stimula-
tion of ethylene biosynthesis by ACC and methylglyoxal
bis(guanylhydrazone) increased SE [110]. Mantiri et al.
[110] reported that, inhibitors of ethylene biosynthesis, it
was shown that ethylene was necessary for SE in M.
truncatula. They demonstrated that SOMATIC EMBRYO
RELATED FACTOR1 (MtSERF1), is induced by ethylene
and expressed in embryogenic calli. RNA interference
knockdown of this gene causes strong inhibition of SE.
This gene is a member of the ethylene response element
(ERF) subfamily based on the classification of Nakano
et al. [121]. Further phylogenetic analysis placed MtSERF1
in Group IX of Nakano et al. [121], which includes the
AtERF5 gene induced by wounding in Arabidopsis [17].
2497
ERF play an important role in hormone signal transduction,
and interconnect different hormone pathways [181]. Eth-
ylene is perceived by a family of five receptors: ethylene
response1 (ETR1), ETR2, ethylene response sensor1
(ERS1), ERS2 and ethylene insensitive4 (EIN4). Genetic
and molecular studies have positioned these receptors
upstream of the mitogen-activated protein (MAP) kinase,
constitutive triple response1 (CTR1), which interacts with
the receptors and also acts as a negative regulator (Fig. 4).
The integral membrane protein, EIN2, and the transcription
factors EIN3 and EIN3-like1 (EIL1) are positive regulators
of ethylene signaling downstream of CTR1. Current mod-
els propose that, hormone binding inactivates the receptors,
resulting in down-regulation of CTR1 activity. Since the
identification of CTR1, biologists have speculated that a
MAP kinase cascade may be involved. Only recently,
however, have putative MAP kinase kinase and MAP
kinase components of the ethylene pathway been identified
[1]. Interestingly, these kinases appear to positively regu-
late ethylene response, suggesting that CTR1 must inhibit
their function. If so, this would represent a novel twist on
the traditional MAP kinase signaling paradigm. Precisely
how the ethylene signal is transduced to the EIN3 and EIL1
transcription factors remains unclear. However, the recent
finding that ethylene stabilizes these transcription factors,
which are targeted for degradation by an SCF complex in
the absence of ethylene, clearly indicates a role for the
ubiquitin pathway [58, 141]. One of the known targets for
EIN3 is the ERF1 transcription factor, which activates
several genes involved in a subset of ethylene responses.
Recently, the ERF transcription factor ERN, required for
nodulation, has been identified in alfalfa [116].
Finding a relationship between an ERF subfamily gene
and the formation of somatic embryos in vitro is consistent
with an emerging picture of the involvement of ERF
transcription factors in developmental processes studied in
vitro.
Stress-related gene and proteins associated
with SE
Plants respond to abiotic stresses by altering the expression
of many of their genes. This altered expression is a major
mechanism of adaptation and survival over the periods of
stress [62]. Early phases of SE are characterized by the
induction of many stress-related genes, which leading to
the hypothesis that SE is an extreme stress response of
cultured plant cells [33]. Stress-related genes were also
induced in these embryogenic cultures, a reason for SE to
be interpreted as the outcome of an in vitro adaptation
process to a new environment [42].
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Fig. 4 A model for ERF1 play

role in ethylene signal
transduction. Ethylene is
perceived by a family of two-
component receptors containing
a consensus or degenerate HK
domain (H). Three of the
receptors also contain a C-
terminal receiver domain (R).
The receptors negatively
regulate ethylene response
together with CTR1 in a
complex on the endoplasmic
reticulum membrane.
Perception results in reducing
receptor and CTR1 activities
and activation of a MAP kinase
kinase, which transmits the
signal through the EIN2
membrane protein, ultimately
the activation of a
transcriptional cascade in the
nucleus

MtSK1 gene
MtSK1 gene is a member of the class of plant kinases
called the SnRK group. Members of the SnRK group of
kinases are thought to play a role in stress responses of
plants. The SnRK protein kinases belong to the recently
categorized CDPK-SnRK superfamily of protein kinases
[71]. The SnRK group was so named because of the
similarity of the kinase domain of these kinases to the
kinase domain of the yeast (Saccharomyces cerevisiae)
SNF1 protein kinase, and to another closely related
protein, the mammalian AMP-activated protein kinase.
Both of these kinases are involved in responses to cel-
lular stresses. The plant SnRK group is further subdi-
vided into three subgroups: SnRK1, SnRK2 and SnRK3,
with the SnRK2 and SnRK3 subgroups apparently being
unique to plants [71]. Most of the SnRK2s characterized
to date are influenced by stresses related to water
availability, and/or respond to the stress-hormone ABA.
Nolan et al. [128] have indicated some subtle changes in
MtSK1 expression in response to ABA in the culture
medium but further work is required to properly inves-
tigate this response. ABA in the culture medium has
been shown to increase the number of embryos that form
on M. truncatula calli, and for this reason 1 lM ABA is
added to the medium after the first 3 weeks of culture
[126]. Also, it has been shown that SnRK2 regulation by
ABA can occur either at the transcription level or at the
protein activation level. For example SnRK2.6 kinase
activity is stimulated by ABA [118, 192], whereas transcript
levels of the SnRK2 genes, serine protein kinase-3
and protein kinases abscisic acid 1 are up-regulated by ABA
[2, 191].
The stress response induced by explant preparation and
culture is increasingly recognized as an important compo-
nent of somatic embryo induction [17]. Santarem et al.
[151] showed that wounding of cotyledons accelerates the
appearance of somatic embryo in soybean. Nolan et al.
[128] have cloned and investigated the stress kinase gene
MtSK1 in relation to SE in M. truncatula. It has been
shown that since elevated levels of MtSK1 expression
occurs in both hormone-containing and hormone-free
media, it is, thus, concluded that its expression is not
dependent on hormonal levels of the culture media.
Excising the tissue up-regulates MtSK1 expression. How-
ever, after the initial wounding response, its expression
continues to increase over several weeks of culture after
which it remains elevated. This may indicate a sustained
stressresponse of cultured tissue throughout the culture
process. The likely role of MtSK1 in stress-induced sig-
naling provides a way forward in relating the stress-
response pathway to the auxin and cytokinin-induced
pathways involved in the induction of SE in the M. trun-
catula culture system.

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GST genes
The transcripts of glutathione-S-transferase (GST) genes
were detected in abundance during auxin induction and in
somatic embryos. GST transcripts have been shown to
accumulate in Chicorium [49], Medicago sativa [172],
Cylcamen [145, 186] and cotton [195] somatic embryos
and GST appears to play a major regulator of the inter-
acting genes sequenced in the present case in response to
auxin. On the other hand, GST genes are induced by ABA
under various biotic and abiotic stresses, and may have a
possible role in detoxifying excessive amounts of auxin,
thus regulating the intracellular concentration or its inac-
tive analogs [57]. Members of the GST gene family are
up-regulated during auxin-induced SE [119, 172] as well as
the initial stages of androgenic development in barley
[182]. Reactive oxygen species (ROS) have been shown to
act as second messenger during auxin and stress-induced
embryogenesis [111, 119]. Nagata et al. [119] found that,
the induction of GST genes during SE is auxin-regulated,
indicating that ROS act as signaling molecules involved in
hormone responses [28, 134]. In agreement with this
hypothesis, increased levels of ROS have been reported to
enhance SE in many plant species [11, 50, 109, 134]. ABA
is capable of increasing H2O2 levels in maize embryos and
seedlings and in Vicia guard cells, further supporting roles
of ROS in ABA signaling [56, 74]. However, molecular
mechanisms mediating ROS production during ABA sig-
naling remain unknown. These findings suggest that the
roles of GST genes during acquisition of embryogenic
potential are likely to be associated with protecting the cell
against the harmful effects of excessive amount of auxin.
Germin-like proteins
Molecular investigations during wheat germination have
revealed unique developmentally regulated proteins, des-
ignated as germins, which show remarkable resistance to
broad specificity proteases and to dissociation in sodium
lauryl sulfate [135]. Germin-like proteins (GLPs) exhibit
sequence and structural similarity with the cereal germins.
Germins/GLPs are apoplastic proteins and part of the cupin
superfamily [35] that includes various proteins identified in
many eukaryotes and prokaryotes. The common feature of
all these proteins is a conserved 3D structure that forms a
six stranded beta barrel [36]. Their localization within the
extracellular matrix and in some cases their hydrogen
peroxide-producing activity suggests that, these proteins
are involved in cell wall metabolism during stress
responses and developmental processes [113]. Some GLPs
such as tobacco Nectarin I [157, 168] are reported to have
oxalate oxidase (SOD) activity. It is interesting in terms of
the physiological role of GLPs that both SOD and oxalate
2499
oxidase (OxO) generate hydrogen peroxide (H2O2) and that
germin and GLPs are located on the apoplast. OxO can
catalyse oxalic acid produced by several plant pathogens
such as Sclerotinia sclerotiorum. OxO can catalyze oxalic
acid produced by several plant pathogens such as Scle-
rotinia sclerotiorum. OxO catalyzes the oxidation of oxalic
acid (stored as calcium salt) by molecular oxygen yielding
CO2, Ca2? and H2O2. OxO also was shown to maintain its
homopentameric structure [99]. To date, the exact mech-
anism by which the OxO is involved in plant defense
responses remains unclear. However, this kind of oxidase
has been implicated as one of the mechanisms for the
generation of ROS during a plant defense response [8].
SOD is considered important in protection of aerobes
against oxidant damage and increased tolerance to oxida-
tive stress is associated with induction of this enzyme. SOD
can dismute superoxide radicals produced by the oxidative
burst. In living cells O2- exists in equilibrium with its
protonated form, the hydroperoxyl radical (O2H). This
radical in either form disproportionates to H2O2 and O2.
This reaction is catalyzed by SOD found in the cytosol,
chloroplasts, and mitochondria [155].
In several cases, GLPs also seem to have non-enzymatic
biochemical activities. They can act as auxin-binding
proteins in peach [130] or serine protease inhibitors in
wheat [157]. Thus, germins/GLPs gene expression is
induced during biotic or abiotic stresses but can also be
related with developmental regulation. In a developmental
context, germin/GLP genes are often expressed during the
early growth stages in wheat embryos [173] and callus
[11], pine [124], Arabidopsis [115], and cotton [9], but also
during organ formation in Arabidopsis [115], barley [34]
and potato [12]. Germins/GLPs could prevent cell expan-
sion by increasing the number of links between polysac-
charides and/or proteins within the cell wall as well as
favouring lignification. Caliskan and Cuming [10]
emphasize that, wheat germins accumulate in cells that
have ceased to expand within the plant but also in auxin-
treated callus cells [11]. The role of apoplastic proteins in
cell differentiation and organogenesis has been extensively
studied in the conifer SE field. Mathieu et al. [113] have
suggested that the GLPs plays a central role in somatic
embryo formation via the regulation of cell wall remod-
eling necessary for correct development in conifers. In
wheat, prior to visible callus formation, there is a striking
induction of germin-like oxalate oxidase gene expres-
sion. Accumulation of gl-OXO mRNA is rapidly stimulated
upon auxin treatment, with a consequent development of
apoplastic enzyme activity producing H2O2 within the cell
wall. Within the dedifferentiated calli, gl-OXO enzyme
activity becomes widespread over the surface of embryo-
genic calli. Differentiation of somatic embryos is initiated
in regions of densely cytoplasmic, meristematic cells that
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are marked by highly localized expression of gl-OXO
activity within these embryogenic cell masses [11]. Vari-
ations in morphology between different embryonic cell
lines have been correlated with differential protein secre-
tion [38]. Such extracellular proteins can even restore
embryogenic capacity to developmentally blocked conifer
cell lines as observed in carrot [106]. In Pinus caribaea,
comparison between the profiles of extracellular proteins of
non-embryogenic and embryogenic cell lines [30] led to
the characterization of the first GLP identified in gymno-
sperms [30]. The cDNA GDP-4-keto-6-deoxy-D-mannose-
3,5-epimerase-4-reductase 1 (GER1) corresponding to this
protein was later isolated in a library and expression
analysis confirmed the embryogenic specificity of this GLP
[124]. Further studies showed that PcGER1 expression was
related to the cell cycle [113] as shown previously in the
case of wheat germin [100]. The PcGER1 gene is unde-
niably associated with cell growth in a heterologous sys-
tem, but little is known about its expression profile during
conifer embryo development. A very similar GLP cDNA,
PrGLP from Pinus radiata was isolated by Bishop-Hurley
et al. [7] after differential screening against non-embryo-
genic tissue transcripts. Global expression analyses con-
firmed that, PrGER1 is expressed in a relatively constant
manner during somatic embryo development in this spe-
cies, but did not provide any indications concerning the
organs or tissues in which the gene is expressed. Never-
theless, this information is essential to understand of the
function of the protein. In wheat, for example, the paradox
of the incompatibility between supposed cell wall cross-
linking activity and the accumulation of the protein in
growing tissues was resolved by demonstrating that a
germin was in fact localized in terminally differentiated
tissues such as coleorhiza, epiblast and scutellum and, later
on, in cells situated beyond proliferating and elongating
cells [10].
Heat shock proteins
In higher plants the heat shock phenomenon was first dis-
covered at the level of protein synthesis in soybean [5, 85].
Heat shock proteins (HSPs) are expressed in response to
increased temperature and other forms of abiotic stress such
as salt, drought, chilling, oxidative stress, and wounding
[17, 29, 185, 194] and facilitate as chaperones the folding
newly synthesized or refolding partially denatured poly-
peptides. The plant HSPs are related to heat shock proteins
of other organisms and to vertebrate alpha-crystalline pro-
teins [26]. All members of the HSP family share a charac-
teristic C-terminal sequence of about 100 amino acid
residues that has also been conserved in the a-crystallin
proteins of the vertebrate eye lens. This sequence is called
the a-crystalline domain, or heat shock-protein domain [47].
123
Mol Biol Rep (2010) 37:24932507
Although it is known that the above described stresses elicit
HSP expression, the molecular mechanisms underlying the
induction and the relationship between heat stress and other
stresses remain unclear. However, in vitro experiments have
demonstrated that some members of HSPs can function as
molecular chaperones in an ATP independent manner [22,
88, 101, 159]. In vivo chaperone function of plant HSPs
was demonstrated by the protection and reactivation of
luciferase in Arabidopsis cells [45, 53]. Members of the
HSP family have been reported during SE [20, 31, 59, 67,
89, 197] as well as initiation of androgenesis by heat and
starvation [4, 23, 164]. These results have led to the
hypothesis that increased levels of HSPs may be associated
with the acquisition of embryogenic potential.
Chitinases
Chitinases are enzymes that hydrolyse b-1,4-N-acetyl-D-
glucosamine (GlcNAc) linkages. Those with lysozyme
activity also cleave b-1,4 linkages between GlcNAc and N-
acetylmuramic acid. An endogenous substrate for plant
chitinases has not yet been found [21]. However, there is
strong evidence that these enzymes catalyse the hydrolytic
decomposition of arabinogalactan proteins (AGPs) present
in plants [179, 180]. Moreover, it is supposed that other
N-acetylglucosamine-containing glycoproteins occurring in
cell walls can be endogenous substrates for plant chitinases
[156, 177]. Expression of chitinase genes can also be
influenced by different stresses [18, 137, 140, 144]. There
are several reports of developmentally regulated chitinase
expression [133]. It was shown that chitinase can stimulate
embryo [3, 25, 39]. It has also been demonstrated that plant
endochitinases expression is spatially and temporally reg-
ulated during plant development processes such as somatic
embryogeneses [25, 26]. Chitinases have also been shown
to stimulate embryo development [4, 178]. Further support
of the involvement of chitinases in developmental pro-
cesses comes from experimental data concerning an
embryogenic suspension culture of the thermosensitive
mutant (ts11) in Daucus carota [25]. SE of the tempera-
ture-sensitive carrot cell mutant ts11 does not proceed
beyond the globular stage at a non-permissive temperature.
The addition of secreted proteins from the wild type D.
carota to ts11 embryo cultures at the nonpermissive tem-
perature appeared to promote the formation of a correctly
formed embryo protoderm. One of the secreted proteins
shown to rescue somatic embryogenesis in the mutant
carrot cell line ts11 was identified as a 32 kDa acidic en-
dochitinase [25]. SE of the temperature-sensitive carrot cell
mutant ts11 does not proceed beyond the globular stage at a
non-permissive temperature. The addition of secreted
proteins from the wild type D. carota to ts11 embryo
cultures at the nonpermissive temperature appeared to
Mol Biol Rep (2010) 37:24932507
promote the formation of a correctly formed embryo pro-
toderm. One of the secreted proteins shown to rescue
somatic embryogenesis in the mutant carrot cell line ts11
was identified as a 32 kDa acidic endochitinase [25]. A
32 kDa acidic endochitinase protein was detected among a
group of extracellular proteins that were found in all stages
of Dactylis glomerata SE [170]. The protein identified in
their studies was an acidic chitinase-like protein that was
constitutively secreted in embryogenic suspension cultures.
Tchorbadjiva and Pantchev [171] also showed that the
accumulation of this protein in the culture medium could
be correlated with the process of D. glomerata SE. Other
data supporting the view that chitinases regulate embryo
development come from experiments with embryogenic
cultures of Cichorium, Picea glauca, and P. abies [3, 32,
37]. The induction of chitinases has also been observed in
the developing zygotic embryos of numerous plants [69,
97]. Wiweger et al. [187] showed that CHIA4-Pa chitinases
promotes proembryogenic masses-to-somatic embryo
transition in Norway spruce. These results imply that
chitinases are involved in early plant somatic embryo
development. The function of chitinases in plant defense
mechanisms is well-known and experimentally proved but
the suggested role of these enzymes in plant growth and
developmental processes require more detailed studies.
b-1,3-Glucanases
b-1,3-Glucanases belong to a large gene family which have
been well characterized in different plant species, particu-
larly in tobacco. They are induced in plants by pathogen
attack or treatment with biotic or abiotic elicitors, and are
also regulated by developmental cues such as changes in
hormonal levels [55, 114]. These b-1,3-glucanases may
have specific functions during normal plant development.
This probably is best illustrated by the role of b-1,3-glu-
canases during pollen formation [9]. Helleboid et al. [63]
have shown that the protein patterns in the explant tissue
and in the medium change and that some of these changes
are associated with the induction and initiation of somatic
embryogenesis in Cichorium. The proteins involved in
these changes were named Somatic embryogenesis related
(SER) proteins. Major proteins such as SER 37.7 kDa,
were isolated from the medium and were identified as
extracellular b-1,3-glucanases [64]. These proteins may be
involved in the degradation of the callose wall surrounding
embryogenic cells and small embryos. It was also shown
that the level of b-1,3-glucanase activity in the medium of
embryo cultures was considerably higher than in medium
from cultures of leaf explants from a non-embryogenic
Cichorium genotype, particularly when embryogenic cells
started to divide and develop into somatic embryos.
Helleboid et al. [65] have also shown the accumulation of
2501
b-1,3-glucanases in the culture-medium conditioned by
somatic embryogenesis initiated in Cichorium 474 leaf
fragments. Expression of b-1,3-glucanases was also
reported during somatic embryogenesis process of spruce
[32] and Araucaria angustifolia [185]. Furthermore, the
accumulation of these 38 kDa b-1,3-glucanases in the
embryo culture medium was reduced when somatic
embryogenesis was inhibited by a-difluoromethylarginine,
an inhibitor of polyamine biosynthesis [63]. These results
suggest that b-1,3-glucanases may has implications in the
somatic embryogenesis process. It is known that the cell
wall around embryonic cells contains callose. The callose
deposition disappears as embryos grow. For this reason, it
has been hypothesized that the culture medium accumu-
lates b-1,3-glucanases which may be responsible for deg-
radation of the callose in the cell wall of embryogenic cells.
The corresponding genes of these enzymes have been
cloned, and their expression supports the hypothesis of a
positive role of this enzyme during somatic embryogenesis
in Chichorium [65].
Conclusions
Somatic embryogenesis is a unique system to investigate
the mechanisms that operate during the transition of a
single somatic cell into an embryogenic entity with the
potential of developing into a complete plant. Stresses are
also the factors that have been recognized as having
important role in the induction of SE. In vitro tissue culture
conditions expose the explants to significant stresses, as
they are removed from their original tissue environment
and placed on synthetic media containing non-physiologi-
cal concentrations of growth regulators. In vitro somatic
embryogenesis is associated with the artificial conditions,
high levels of exogenous growth regulators and many other
stress factors. Furthermore, embryogenic competence
somatic cells can stimulate by various factors, such as
heavy metal ions, high osmotic pressure, dehydration,
explant wounding and high temperature. These stressful
conditions may result in a general stress response in cells
showing extended chromatin reorganization. Early phases
of SE are characterized by the induction of many stress-
related genes, which leads to the hypothesis that SE is an
extreme stress response of cultured plant cells. Despite the
progress achieved during the last few years in under-
standing the muscular mechanisms involved in SE, there
are still many aspects that are not fully understood and
need to be studied in more detail. Future research in this
area must center not only on isolating and characterizing
large numbers of genes expressed in early phases of SE, but
also on deciphering the significance of these genes by
demonstrating what happens when their function is
123
2502
disrupted. This is being attempted either by creating
transgenic plants that express an antisense construct or by
working with genes whose functions have already been
changed through gain- and loss-of-function mutations.
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