Plant Cell Monogr (2)
A. Mujib · J. Šamaj: Somatic Embryogenesis
DOI 10.1007/7089_040/Published online: 30 November 2005
© Springer-Verlag Berlin Heidelberg 2005
Mode of Action
of Plant Hormones and Plant Growth Regulators
During Induction of Somatic Embryogenesis:
Molecular Aspects
Clément Thomas1 (
) · Víctor M. Jiménez2
1Plant Molecular Biology, CRP-Santé, Bâtiment modulaire,
84 Val Fleuri, 1526 Luxembourg, Luxembourg
clement.thomas@crp-sante.lu
2CIGRAS, Universidad de Costa Rica, 2060 San Pedro, Costa Rica
vjimenez@cariari.ucr.ac.cr
Abstract Plant hormones play critical roles in the establishment of somatic embryogene-
sis. During this process, somatic plant cells reverse their state of differentiation, acquire
pluripotentiality and set up a new developmental program. The identification of the
regulatory mechanisms that govern the key events of somatic embryogenesis requires mo-
lecular and genetic investigations. One critical issue is how plant hormones and growth
regulators act to mediate somatic embryogenesis. Do they function as simple stimuli or
participate directly, as central signals, in the reprogramming of the somatic cells towards
an embryogenic fate? The latter scenario is now well supported by a number of studies
that provide evidence of close interconnections between plant hormones and the molecu-
lar pathways that control somatic embryogenesis, including chromatin remodeling, gene
expression patterning, reactivation of cell cycle and division and regulation of protein
turnover. In this chapter we describe recent advances in the understanding of molecular
and genetic mechanisms underlying the early stages of somatic embryogenesis. The roles
and mode of action of plant hormones are especially emphasized.
Abbreviations
2,4-D 2,4-Dichlorophenoxyacetic acid
ABA Abscisic acid
ABP1 Auxin binding protein 1
ARF Auxin-response factors
aza-C 5-Azacytidine
BBM BABY BOOM
BAP Benzylaminopurine
CDK Cyclin-dependent kinase
DD-RT PCR Differential display reverse transcription polymerase chain reaction
ER Endoplasmic reticulum
GA Gibberellin
IAA Indole-3-acetic acid
LEC LEAFY COTYLEDON
NAA Naphthalene acetic acid
PGR Plant growth regulator
158 C. Thomas · V.M. Jiménez
PH Plant hormone
PKL PICKLE
SE Somatic embryogenesis
(SERK) SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE
(WUS) WUSCHEL
1
Introduction
Somatic embryogenesis (SE) has been observed to be induced by different
factors (reviewed by Jiménez 2001; Fehér et al. 2003). Independently of the
nature of the external stimulus, the establishment of SE necessarily involves
profound changes at the molecular level, such as the coordinated expression
of different sets of genes that drive the switch from the current vegetative
growth pattern to an embryogenic development. Thus, the identification of
the genes that trigger key phases of SE, i.e. cell dedifferentiation, cell cycle
reentry and establishment of a new embryogenic fate, is highly desirable.
Additionally, the elucidation of the signaling pathways by which plant cells
remodel their gene expression program is central to understanding the reg-
ulation of the SE process.
As discussed in detail elsewhere (Jiménez and Thomas, this volume), plant
growth regulators (PGRs) are among the external stimuli most often em-
ployed to induce SE and to regulate the further development of embryogenic
tissues. There was some controversy as to whether PGRs/plant hormones
(PHs) act only as stimuli or are more directly involved in the mechanisms
that regulate gene expression (Gaspar et al. 2003). However, during the last
few years, a large body of experimental data supports the view that PHs play
a central role in the establishment of SE. The understanding of the underly-
ing mechanisms of PH action requires investigation of hormone receptors,
signal transduction pathways, and genetic programs that lead to the final cell
response.
The first step in any event associated with a response to a hormone is
a proper recognition by the target cells. This recognition normally involves
receptors, which are proteins associated with the cell membranes or are lo-
cated in the cytoplasm. Receptors have been identified and characterized for
hormone groups such as ethylene (Chang et al. 1993; Schaller and Bleecker
1995) and cytokinins (Inoue et al. 2001; Ueguchi et al. 2001). These receptors
activate a signal transduction pathway that either induces or inhibits cellular
functions, or controls gene expression (reviewed by Kulaeva and Prokoptseva
2004).
In the case of auxins, although some auxin-binding proteins have been
isolated, it is still uncertain whether they represent receptors for different
auxin-mediated processes (Gaspar et al. 2003). To date, auxin binding pro-
Plant Hormones, Molecular Aspects 159
tein 1 (ABP1) is the best-studied putative auxin receptor protein. ABP1 pre-
dominantly accumulates in the lumen of the endoplasmic reticulum (ER), an
unusual location for a hormone receptor (Barbier-Brygoo et al. 1989; Inohara
et al. 1989). However, there is evidence that ABP1 is active in auxin respon-
siveness at the surface of the plasma membrane although it carries the ER
retention motif (Rück et al. 1993; Thiel et al. 1993; Leblanc et al. 1999; Stef-
fens et al. 2001; Shimomura et al. 1989). ABP1 has been shown to mediate
early auxin responses such as auxin-induced electrical responses (Rück et al.
1993; Thiel et al. 1993; Zimmermann et al. 1994; Bauly et al. 2000) and cell ex-
pansion (Jones et al. 1998; Chen et al. 2001a, b). However, its involvement in
auxin-induced gene expression has not been proved yet. Although mechan-
isms responsible for auxin signal transduction from receptor to genome are
still poorly known, significant progress has been achieved in auxin-regulated
gene expression (reviewed by Hagen and Guilfoyle 2002).
The PGRs most widely used to induce and regulate in vitro SE are aux-
ins and cytokinins. It has been observed that members from both hormone
groups regulate the cell cycle and trigger cell divisions (Francis and Sorrell
2001), two very important factors that have been related to initiation of SE
(Dudits et al. 1991, 1995; Yeung 1995). Recent data provide evidence that the
elaboration and execution of developmental programs require a proper con-
trol of the cell cycle and division, indicating the regulators of the cell cycle
machinery as key determinants of SE.
In addition to their influence on cell cycle progression, PGRs/PHs have
been demonstrated to trigger substantial changes in chromatin structure and
alteration of transcription that lead to the formation of either dedifferentiated
callus tissues or somatic embryos (Dudits et al. 1995). Studies on the links be-
tween PH action and gene expression have resulted in the cloning of several
genes responsive to auxins, cytokinins, or to both hormones. Although, the
functions of a number of these genes remain unknown, others have obvious
connections with the cell cycle or developmental processes including SE.
In this chapter we describe those findings related to cell division and
changes in the pattern of gene expression during early stages of SE and we
further highlight how hormonal signals are integrated into these processes.
2
Reactivation of Cell Cycle and Division
The reactivation of the cell cycle and division in differentiated cells is in-
dispensable for the initiation of plant developmental processes, including SE
(Dudits et al. 1995). The cell cycle is usually divided in four sequential phases:
G1, S (DNA replication), G2 and M (cytokinesis). The basic control mech-
anisms that regulate progression through the cell cycle are remarkably well
conserved during evolution and operate mainly at the G1–S and G2–M tran-
160 C. Thomas · V.M. Jiménez
sitions (reviewed by Stals and Inzé 2001; De Veylder et al. 2003; Dewitte
and Murray 2003). These two key check points depend on highly conserved
serine/threonine kinases, named cyclin-dependent kinases (CDKs), and their
associated regulatory subunits called cyclins.
The two mentioned groups of hormones, auxin and cytokinins, are gen-
erally sufficient to stimulate and sustain the in vitro proliferation of most
plant cell types and have therefore been the best documented direct regula-
tors of the cell cycle progression. The expression of genes related to the cdc2
gene, which encodes the catalytic subunit of the key G1–S and G2–M regula-
tor cdc2 protein kinase, is upregulated by auxin in alfalfa (Hirt et al. 1991),
soybean (Miao et al. 1993) and tobacco pith explants (John et al. 1993). Al-
though auxin enhances cdc2 gene expression, cotreatment with cytokinin is
absolutely required to induce a basic cdc2 expression in tobacco pith explants
(John et al. 1993), illustrating the synergic regulation exerted by both growth
regulators on cell proliferation. The observation that most systems require
only exogenously added auxin to resume cell division suggests that the rate
of endogenous cytokinin synthesis is sufficient to sustain growth (del Pozo
et al. 2005). In situ analysis of cdc2 expression in plants such as Arabidop-
sis (Martinez et al. 1992; Hemerly et al. 1993) and soybean (Miao et al. 1993)
revealed that cdc2a expression is not only associated with cell proliferation
but also precedes it, suggesting that it reflects a state of competence to divide
(Hemerly et al. 1993).
More recently, auxin has been shown to upregulate the expression of an al-
falfa A2-type cyclin, whose promoter contains auxin-response-like elements
(Roudier et al. 2003). In addition, auxin treatment of alfalfa plants affects the
spatial expression pattern of this cyclin by shifting its expression from the
phloem to the xylempoles, where lateral root formation is initiated in response
to auxin. This auxin-regulated spatial cyclin expression illustrates another
aspect of the complexity of hormonal regulation of the cell cycle in planta.
Cyclins D represent important connections between PHs and the cell cycle.
Consistent with its regulatory function in the cell cycle progression, cyclin
CycD3 is expressed in tissues having a high rate of cell divisions, including
shoot meristems, young leaf primordia, axillary buds, procambium and vas-
cular tissues of developing leaves (Riou-Khamlichi et al. 1999). The CycD3
gene is highly responsive to cytokinin in both cell cultures and whole plants
and is rapidly induced by cytokinin during the G1 phase of cells reentering
the cell cycle. Constitutive expression of this cyclin in transgenic Arabidopsis
plants leads to diverse disorders, e.g., extensive leaf curling and disorga-
nized meristems, and, importantly, it renders callus growth independent of
cytokinin application (Riou-Khamlichi et al. 1999). This demonstrates that
cytokinins promote cell division by inducing the CycD3 expression at the
G1–S phase transition.
Cytokinins have also been reported to play a regulatory role at the mitotic
control point of the G2–M transition. This is well illustrated by the observa-
Plant Hormones, Molecular Aspects 161
tion that application of the cytokinin biosynthesis inhibitor lovastatin blocks
cells of tobacco BY-2 in G2 (Laureys et al. 1998). This effect is nullified by the
addition of an exogenous cytokinin, such as zeatin. This is in line with pre-
vious observations that cytokinins accumulate transiently during the G2–M
phase (Redig et al. 1996) and that the removal of cytokinin from the culture
medium leads to an arrest in G2 of tobacco suspension cell cultures, which
accumulate inactive CDK complexes (Zhang et al. 1996). The kinase activity
of the latter is restored by either addition of cytokinin or by tyrosine de-
phosphorylation, suggesting that the inactivation of CDK complexes under
cytokinin deprivation is due to phosphorylation of regulatory residues of the
CDK subunit.
Although auxin and cytokinins are generally considered as the main hor-
monal signals triggering cell cycle progression, others PHs with enhancing
or inhibitory functions participate in the cell cycle control by modulating
the transcriptional expression of different cell cycle genes. For example, gib-
berellin (GA) stimulates CDK and cyclin accumulation in a tissue-specific
manner (Sauter 1997). Abscisic acid (ABA) induces a decrease in Cdc2a-like
kinase activity by increasing the expression level of a CDK inhibitor gene,
namely the ICK1 gene, whose product interacts with Cdc2a and CycD3 (Wang
et al. 1998). Although its mode of action is still unclear, jasmonic acid has
been reported to block synchronized BY2 cells in both G1–S and G2–M tran-
sitions (Swiatek et al. 2002).
3
Reprogramming of the Gene Expression Pattern
The establishment of totipotency and the subsequent induction and devel-
opment of somatic embryos require reprogramming the cultures. This is in
part achieved by synthesis of new RNA molecules. Therefore, early induc-
tive molecular events have been investigated by monitoring gene transcripts
that are synthesized under the influence of external stimuli that trigger the
embryogenic fate. Several examples of changes in the expression of genes re-
lated to initiation of SE have been reported. Here, we make reference only to
those works in which the change in gene expression can be traced back to
PGRs/PHs.
In an attempt to identify genes that switch on the SE program, researchers
have employed systematic approaches aimed at comparing the population
of transcripts expressed in embryogenic conditions with the population of
the transcript expressed in nonembryogenic conditions. This was carried out
using techniques such as differential complementary DNA library screening,
differential display reverse transcription polymerase chain reaction (DD-RT
PCR), cold plaque screening and more recently microarrays. The application
of these techniques to the induction phase of SE has been complicated by the
162 C. Thomas · V.M. Jiménez
difficulty to identify and isolate embryogenic cells in the initial steps when
no morphological changes are visible. However, improvements in in vitro cul-
ture systems and methods of molecular analysis have allowed progress in the
exploration of the early phases of SE.
Nagata et al. (1994) isolated three auxin-regulated genes, parA, parB and
parC, that are transiently expressed during the regaining of meristematic ac-
tivity of tobacco mesophyll protoplasts. The corresponding transcripts were
detected as early as 20 min after the beginning of incubation of protoplasts
with auxin. Importantly, they were no longer detected after 48 h of culture
when protoplasts started to divide, suggesting that they are specifically in-
volved in the reentry into the plant cell cycle.
Kitamiya et al. (2000) isolated two carrot genes that are differentially ex-
pressed in hypocotyl cells induced to form somatic embryos by treatment
with 2,4-dicholorophenoxyacetic acid (2,4-D) for 2 h. One of these genes,
namely the D. carota heat-shock protein 1 (Dchsp-1) gene, is related to low
molecular weight heat-shock proteins and was found to be expressed during
embryo development. The other gene has homology to the auxin-regulated
genes, including par A (Takahashi et al. 1989), and thus was named D. carota
auxin-regulated gene 1 (Dcarg-1). Interestingly, there is a parallel relationship
between the expression of Dcarg-1 and the formation of somatic embryos.
In addition, in contrast to Dchsp-1, Dcarg-1 was not responsive to stress
treatment and was not expressed during development of somatic embryos,
implying that its function was not required for this process to occur.
Using DD-RT PCR, Yasuda et al. (2001) attempted to identify genes that are
preferentially expressed during the early stages of auxin-induced carrot SE.
Three transcripts that accumulate immediately after somatic cells divide to
form cell clusters, but that do not accumulate or barely accumulate in nonem-
bryogenic cell suspension cultures, were characterized. Although these genes
represent potential key regulators of SE, a clear function has still not been
attributed to them.
An important issue is how PH action on the gene expression level pattern
is mediated. In a general view, the hormonal signal activates a signaling cas-
cade that recruits specific transcription factors. These induce the expression
of target genes, which in turn trigger the final response. Numerous genes have
been described containing cis-acting elements in their promoter region that
confer hormone responsiveness. Over the past 20 years, sequences that are
upregulated or downregulated by PHs have been described for auxins (Guil-
foyle et al. 1998; Ulmasov et al. 1999), ABA (Marcotte et al. 1992), GAs (Gubler
and Jacobsen 1992) and ethylene (Meller et al. 1993).
The auxin-modulated gene expression system is based, at least in part,
on two interacting protein families. The multifamily protein auxin-response
factors (ARFs) can activate or repress target genes by directly binding to spe-
cific DNA sequences, i.e., auxin response elements (Ulmasov et al. 1999). In
contrast, the auxin/indole-3-acetic acid (IAA) proteins do not bind to DNA
Plant Hormones, Molecular Aspects 163
directly but can inactivate ARF transcription factors by interacting with them
through heterodimerization (Tiwari et al. 2001). Auxin exerts its regulation
on gene expression through modulation of auxin/IAA protein turnover via
a specialized branch of the ubiquitin–proteasome pathway (Worley et al. 2000;
Gray et al. 2001; Dharmasiri and Estelle 2002). Such a PH-regulated proteol-
ysis has been shown to be involved in many aspects of plant developmental
processes including SE.
4
Chromatin Structure and DNA Methylation
A specific gene expression program is the result of the balance between the
part of the genome that is transcribed, i.e., euchromatin, and the part that is
repressed, i.e., heterochromatin. Many aspects of plant development, includ-
ing embryonic and meristem development, flowering and seed formation,
involve modifications of chromatin structure that affect the accessibility of
target genes to regulatory factors that control their expression (reviewed by
Li et al. 2002). Since maintaining the cellular differentiated state largely relies
on chromatin-dependent gene silencing, the cellular dedifferentiation and the
switch to a new embryogenic program necessarily involve important changes
in chromatin structure.
Zhao et al. (2001) identified two distinct phases of chromatin decondensa-
tion during in vitro induced dedifferentiation of tobacco mesophyll cells. The
first was independent of any hormonal treatment and was linked to the acqui-
sition of pluripotentiality or dedifferentiation of cells. In contrast, the second
phase of chromatin decondensation required auxin and cytokinin treatment
and was linked to the reentry into the S phase.
Dynamic changes in chromatin structure are influenced by both posttrans-
lational modifications of histone amino terminal tails and direct modifica-
tions of the DNA, such as methylation. The degree of DNA methylation has
been reported to influence plant morphogenesis (reviewed by Li et al. 2002).
The overexpression of an antisense DNAmethyltransferase copy in transgenic
tobacco plants provokes development disorders, including small leaves, short
internodes and abnormal flower morphology (Nakano et al. 2000). The role
of DNA methylation in early phases of SE has been recently addressed by Ya-
mamoto et al. (2005) by investigating the effects of 5-azacytidine (aza-C), an
inhibitor of DNA methylation, on the induction of direct carrot SE. Aza-C
treatment totally inhibited the formation of embryogenic cell clumps from
epidermal carrot cells. When applied during morphogenesis of embryos,
aza-C downregulated the expression of C-LEC1, an important gene that par-
ticipates in the embryonic program (Sect. 6). Additionally, in untreated cells,
a DNA methyltransferase gene transcript transiently accumulated after auxin
application but before the formation of embryogenic cell clumps, suggest-
164 C. Thomas · V.M. Jiménez
ing a direct role for DNA methylation in the establishment of embryogenic
competence in carrot somatic cells.
Other chemical substances, such as the antibiotic kanamycin, have been
observed to considerably modify the level of DNA methylation during plant
in vitro culture (Bardini et al. 2003). In this case, DNA methylation is con-
sidered as a potential source of somaclonal variation, a phenomenon (often
undesirable) observed in plant cell and tissue cultures (Caplan et al. 1998).
5
Some Key Regulators of the Vegetative-to-Embryogenic Transition
As already stated, different strategies have been used to identify genes that
are differentially expressed during SE (Thomas 1993; Lin et al. 1996; Schmidt
et al. 1997). Although several genes have been cloned, their function or func-
tions often remain obscure. However, improvements in plant transformation
protocols and the availability of new mutants allowed the characterization
of genes that regulate the vegetative-to-embryogenic transition. The ectopic
expression of these genes either enhances SE in in vitro cultures or even pro-
vokes spontaneous embryo formation on intact plants. One new challenge is
to identify possible existing links between the PRG/pH and the genes that
possibly influence the vegetative-to-embryogenic transition during SE.
5.1
The SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE Gene
Most of the molecular markers of SE identified to date are related to late
stages of embryo development. However, one gene, encoding a leucine-rich-
repeat receptor-like kinase, has been found to be specifically upregulated
during the very precocious phases of the SE process. The SOMATIC EMBRYO-
GENESIS RECEPTOR-LIKE KINASE (SERK) gene was originally cloned from
a carrot cell suspension culture where it was found to mark cells that are com-
petent to form somatic embryos, i.e., cells in transition between the somatic
and the embryogenic states (Schmidt et al. 1997). Using in situ hybridization,
DcSERK expression was shown to first appear in single cells of embryogenic
cultures induced with 2,4-D for 7 days. DcSERK expression continues until
the 100-cell stage of the globular somatic embryo and then ceases. Interest-
ingly, a similar SERK expression pattern was observed during early zygotic
embryogenesis, suggesting that the same SERK signaling pathway is activated
during both SE and zygotic embryogenesis (Schmidt et al. 1997).
Several homologs of the carrot DcSERK have been identified in mono-
cots, e.g., maize (Baudino et al. 2001) and Dactylis glomerata (Somleva et al.
2000), and dicots, e.g., Medicago truncatula (Nolan et al. 2003), Arabidop-
sis thaliana (Hecht et al. 2001) and sunflower (Thomas et al. 2004). Plant
Plant Hormones, Molecular Aspects 165
genomes contain several SERK genes. As an example, the Arabidopsis SERK
gene family comprises five members (Hecht et al. 2001). The expression of
AtSERK1, the Arabidopsis gene most closely related to the carrot DcSERK,
also marks embryogenic competent cells in culture. As in carrot, the level of
SERK expression increases in response to the auxin treatment used to induce
somatic embryos. Auxin-dependent SERK expression was also reported for
the M. truncatula MtSERK gene, which is upregulated by the auxin naphtha-
lene acetic acid (NAA) but not by the cytokinin benzylaminopurine (BAP;
Nolan et al. 2003). However, addition of BAP to the culture medium poten-
tiates NAA-induced SERK expression, possibly by stimulating endogenous
auxin synthesis. In the direct SE system of sunflower, SERK transcripts spe-
cifically accumulate in the future morphogenic region of explants within the
first few hours of culture. Although the only PGR supplied in the medium is
a cytokinin, analysis of the endogenous PH content revealed that the internal
IAA concentration transiently increases in explants during this early period
(Thomas et al. 2002). A link between auxin and SERK expression is also sug-
gested by the accumulation of SERK transcripts in plant tissues that contain
high auxin levels, e.g., vascular tissue and leaf primordia (Hecht et al. 2001;
Thomas et al. 2004). However, since SERK is not induced by auxin in all the
cell explants or cell cultures, it is probably not an integral part of the auxin
machinery or its expression requires other, still unknown, factors (Hecht et al.
2001).
Evidence that AtSERK1 is not only a good marker of embryogenic com-
petent cells in Arabidopsis but is also involved in the establishment of the
embryogenic competence comes from ectopic overexpression of the AtSERK1
gene in Arabidopsis (Hecht et al. 2001). Although during normal growth
transgenic seedlings do not show any specific phenotype, their embryogenic
capacity is considerably enhanced (approximately 4 times compared with the
wild type) during in vitro culture. A similar increase in embryogenic com-
petence is conferred by mutation in shoot apical meristem regulatory genes
such as AMP1, CLV1 and CLV2 (Mordhorst et al. 1998). The higher AtSERK1
expression level in amp1 cultures, in comparison with that in wild-type cul-
tures, suggests that one role of AMP1 could be to downregulate the expression
of AtSERK1 after germination (Hecht et al. 2001).
The identification of SERK-activating ligand(s) as well as the downstream
targets of SERK is highly desirable to further characterize the function(s) of
SERK in both zygotic embryogenesis and SE.
5.2
The BABY BOOM Gene
Another gene that potentially activates signal transduction pathways lead-
ing to the induction of embryo development from differentiated somatic cells
is the BABY BOOM (BBM) gene (Boutilier et al. 2002). It was identified
166 C. Thomas · V.M. Jiménez
by a screening approach aimed at identifying genes differentially expressed
during early phases of Brassica napus microspore embryogenesis. The B. na-
pus microspore culture system relies on the ability of the vegetative cell of
an immature pollen grain to develop into an embryo in response to high-
temperature (above 25 ◦C) culture conditions (Custers et al. 1994).
The BBM gene encodes a protein that belongs to the AP2/ERF family,
a plant-specific class of transcription factors that regulate several develop-
mental processes, such as floral organ identity determination and control of
leaf epidermal cell identity (reviewed by Riechmann and Meyerowitz 1998).
It is preferentially expressed during embryo and seed development (Boutilier
et al. 2002).
Overexpression of the BBM gene under the control of a constitutive pro-
moter leads to the spontaneous formation of somatic embryos and cotyledon-
like structures on different tissues of intact plants (Boutilier et al. 2002).
Additionally, in vitro cultured explants, coming from BBM-overexpressing
transgenic plants, display an enhanced capacity to regenerate through shoot
organogenesis. This suggests that BBM plays a broader role in cell division
and differentiation rather than being a specific element of the SE pathway.
Importantly, in contrast to SERK, ectopic expression of BBM is able to pro-
mote SE in the absence of exogenously applied PGR. It has been proposed that
BBM could act by stimulating an increase of PH and/or increasing the cellular
hormonal sensitivity (Boutilier et al. 2002). In that sense, Klucher et al. (1996)
speculated that AP2/ERF domain proteins, being unique to plants, might
have coevolved with plant-specific pathways such as PH signal transduction.
Alternatively, it is also conceivable that the BBM product acts in a PH signal-
ing pathway downstream of the hormone perception as previously shown for
some other AP2/ERF domain proteins (Finkelstein et al. 1998; Menke et al.
1999; Gu et al. 2000; Banno et al. 2001; van der Fits and Memelink 2001).
5.3
The LEAFY COTYLEDON Genes
Arabidopsis mutants that display abnormalities in embryo development rep-
resent powerful tools to investigate the molecular pathways underlying SE.
The LEAFY COTYLEDON1 (LEC1) and LEAFY COTYLEDON2 (LEC2) genes
were identified originally as loss-of-functionmutants showing defects in both
embryo identity and seed maturation processes (Meinke et al. 1994; West
et al. 1994). Lec embryos present a reduction in desiccation tolerance and
do not accumulate normal storage materials. In addition, lec mutants ex-
hibit other anatomical characteristics, including the presence of trichomes on
cotyledons, which in Arabidopsis wild-type plants are specific to true leaves
(Meinke et al. 1994; West et al. 1994; Stone et al. 2001). The pleiotropic effects
of lec mutations pinpoint the LEC genes as central regulators of embryo and
seed development.
Plant Hormones, Molecular Aspects 167
Identification and analysis of the Arabidopsis LEC1 and LEC2 genes con-
firmed their regulatory roles in embryogenesis and provided significant in-
sight into their functions. Both LEC genes encode seed-expressed transcrip-
tional activators. LEC1 encodes a protein related to the heme-activated pro-
tein 3 subunit of the CCAAT box-binding factor, a eukaryotic transcription
factor (Lotan et al. 1998). LEC2 encodes a protein that contains the plant-
specific B3 domain (Stone et al. 2001), which is found in several plant tran-
scription factors including ABA INSENSITIVE3 (Luerssen et al. 1998) and
VIVIPAROUS1 (McCarty et al. 1989 and 1991).
Ectopic overexpression of either lec1 or lec2 results in the spontaneous for-
mation of somatic embryos directly on the leaf surface, suggesting that lec
genes play a role in conferring embryogenic competence to cells (Lotan et al.
1998; Stone et al. 2001). It also confers embryonic characteristics to seedlings.
The expression of embryo-specific genes, such as those encoding cruciferin
A, 2S storage protein and oleosin, in adult transgenic seedlings, confirms the
activation and maintenance of embryo-specific programs in vegetative tis-
sues. Interestingly, the 35S::LEC1 phenotype is relatively weak, i.e., only a few
plants show sporadic embryo development, whereas the 35S::LEC2 phenotype
is stronger and comparable to that observed for 35S::BBM plants. The fact that
the BBM and LEC genes exhibit similar putative functions as transcription
factors, are both preferentially expressed in seeds, and confer to plants a simi-
lar phenotype when ectopically expressed suggests that they function in the
same molecular pathway. However, BBM transcripts are present in lec1 mu-
tant seeds (Boutilier et al. 2002), indicating that the expression of BBM is not
dependent per se on the presence of the LEC1 protein. Thus, BBM could either
function upstream of LEC1 or operate in an LEC1-separated but overlapping
pathway.
Recently, Yazawa et al. (2004) isolated a carrot functional homolog of Ara-
bidopsis LEC1, as demonstrated by complementation experiments. In the
SE system of carrot, the highest expression of C-LEC1 was detected in cell
clusters of 38–63 µm in diameter that were being cultured for induction of so-
matic embryos. Strikingly, cell clusters of this size are also those that are the
most efficient for somatic embryo production (Satoh et al. 1986).
5.4
The PICKLE Gene
Another interesting Arabidopsis mutant is the pickle (pkl) mutant described
by Ogas et al. (1997). At the opposite side of the lec phenotypes, a null mu-
tation in the PKL gene induces embryonic characteristics in the roots of
Arabidopsis seedlings, including accumulation of lipids and seed storage pro-
teins normally found in seeds (Ogas et al. 1999; Rider et al. 2004). When
excised and cultured on a medium lacking PGR, roots of pkl seedlings sponta-
neously develop somatic embryos. Exogenous application of GA is sufficient
168 C. Thomas · V.M. Jiménez
to suppress the mutant phenotype, whereas decreasing the level of GA in
germinating seeds increases significantly the penetrance of the pkl root phe-
notype (Ogas et al. 1997). These observations suggest that PKL functions
in a GA pathway that controls the switch of root cells from an embryonic
to a vegetative fate. The PKL gene encodes a CHD3-chromatin remodeling
factor, and thus is likely to function as a negative regulator of transcrip-
tion of embryo-specific genes (Eshed et al. 1999; Ogas et al. 1999). This
is supported by the observation that LEC1 and LEC2 expression levels are
significantly higher in pkl than in wild-type seedlings. Thus, expression of
embryonic traits in pkl seedlings is highly suspected to be a consequence of
the failure to repress expression, in a GA-dependent manner, of the master
regulators of embryogenic identity, such as the LEC genes, during germi-
nation (Rider et al. 2003). However, as noted by Henderson et al. (2004),
data that demonstrate a direct link between PKL activity and GA are still
missing and thus it could not be absolutely decided whether repression
of LEC1 is or is not a GA-dependent event. The observation that GA can
act in the absence of PKL to repress expression of the pkl root phenotype
(Ogas et al. 1997) demonstrates that there also exists a PKL-independent
pathway by which GA represses expression of embryonic traits. This is
consistent with the recent metabolic analysis that revealed that pkl Ara-
bidopsis roots accumulate some but not all seed-specific metabolites (Rider
et al. 2004).
5.5
The WUSCHEL Gene
Using a genetic approach to identify gain-of-function mutations that can pro-
mote embryogenic callus formation fromArabidopsis root explants, Zuo et al.
(2002) identified a gene, PAG6, that was found to be identical to WUSCHEL
(WUS), a gene previously characterized as a key regulator for specification of
stem cell fate in floral and shoot meristems (Laux et al. 1996). WUS encodes
a homeodomain protein and is expressed in a small group of cells, namely, the
organizing center, below the shoot meristem central zone, which contains the
stem cells (Mayer et al. 1998; Schoof et al. 2000).
Overexpression of WUS induces the formation of highly embryogenic cal-
lus in the presence of auxin (Zuo et al. 2002). In addition, ectopic over-
expression of WUS in transgenic plants directly induces somatic embryos
from various vegetative tissues independently of any external PGR treatment.
Therefore, WUS appears to be able to trigger the vegetative-to-embryogenic
transition, bypassing the auxin requirement or taking advantage of the en-
dogenous auxin flux (Zuo et al. 2002).
Interestingly, WUS cannot reprogram the shoot apex towards SE when
overexpressed under the control of meristem-specific promoters such as
CLV1, ANT (Schoof et al. 2000), LFY, AP3 and AG (Lenhard et al. 2001;
Plant Hormones, Molecular Aspects 169
Lohmann et al. 2001). This raises the possibility that some factors could favor
one or the other WUS function (a shoot meristem or an embryo organizer).
Gallois et al. (2004) addressed this possibility by studying the effects of ec-
topic expression of WUS in roots. In the absence of additional cues, WUS
expression in the root induced shoot stem identity and leaf development
indicating that WUS establishes stem cells with an intrinsic shoot identity.
However, when WUS is coexpressed with LEAFY, which is a master regula-
tor of floral development (Weigel et al. 1992), WUS induces the formation of
floral tissues. Finally, when exogenous auxin is supplied, the expression of
WUS leads to the development of somatic embryos. This elegant work demon-
strates that although WUS expression specifies an intrinsic shoot activity (in
the absence of additional cues) it also makes cells developmentally flexible
and able to be directed to floral organ or embryo development, depending on
additional cues.
6
Concluding Remarks
PGRs/PHs are largely used to elicit in vitro SE and are therefore suspected to
play important roles in this process; however, the question of their exact func-
tion remains open. One difficulty in elucidating the role of PGRs/PHs in SE is
that they are likely to be involved at different levels. Although they are very
efficient stimuli, they also represent signaling molecules that are an integral
part of the molecular pathways underlying SE. As exogenous stimuli, they
can occasionally be replaced by other treatments, including stresses such as
osmotic or heat shock (Jiménez and Thomas, this volume). In contrast, it be-
comes obvious that endogenous PHs play essential roles in directing crucial
SE-related events, including reentry into the cell cycle and dedifferentiation
and redifferentiation of somatic cells. Recent developments in the elucidation
of modes of action of PHs have shown that they trigger profound modifi-
cations in cellular gene expression patterns both by influencing chromatin
structure and DNA methylation and by a finer and more specific transcrip-
tional regulation of target genes.
Recent data suggest that the cellular embryonic competence is “actively”
repressed in postembryonic plant tissues by proteins such as AMP1 or
PICKLE. Derepression, e.g., by null mutation in repressor genes, opens the
way to SE. However, somatic embryo induction is only activated when local
tissue/cellular conditions, such as a proper hormonal balance, are appro-
priate. This would explain why all cells of pickle or amp1 mutants do not
uniformly enter an embryonic developmental program. The observation that
different mutations induce similar embryonic phenotypes in postembryonic
plants reflects the complexity of SE and the possible existence of overlapping
pathways triggering this developmental process.
170 C. Thomas · V.M. Jiménez
In recent years functional genomics allowed identification of several po-
tential candidate genes that may be responsible for the establishment of the SE
program. Although the participation of these genes in the induction of SE in
wild-type plants has not been proved yet, they represent very exciting tracks
to pursue in the exploration of molecular pathways underlying SE.
References
Banno H, Ikeda Y, Niu QW, Chua NH (2001) Overexpression of Arabidopsis ESR1 induces
initiation of shoot regeneration. Plant Cell 13:2609–2618
Barbier-Brygoo H, Ephritkhine G, Klämbt D, Ghislain M, Guern J (1989) Functional ev-
idence for an auxin receptor at the plasmalemma of tobacco mesophyll protoplasts.
Proc Natl Acad Sci USA 86:891–895
Bardini M, Labra M, Winfield M, Sala F (2003) Antibiotic-induced DNA methylation
changes in calluses of Arabidopsis thaliana. Plant Cell Tissue Organ Cult 72:157–162
Baudino S, Hansen S, Brettschneider R, Hecht VF, Dresselhaus T, Lörz H, Dumas C, Ro-
gowsky PM (2001) Molecular characterisation of two novel maize LRR receptor-like
kinases, which belong to the SERK gene family. Planta 213:1–10
Bauly JM, Sealy IM, Macdonald H, Brearley J, Droge S, Hillmer S, Robinson DG, Ve-
nis MA, Blatt MR, Lazarus CM, Napier RM (2000) Overexpression of auxin-binding
protein enhances the sensitivity of guard cells to auxin. Plant Physiol 124:1229–1238
Boutilier K, Offringa R, Sharma VK, Kieft H, Ouellet T, Zhang L, Hattori J, Liu CM, van
Lammeren AA, Miki BL, Custers JB, van Lookeren Campagne MM (2002) Ectopic ex-
pression of BABY BOOM triggers a conversion from vegetative to embryonic growth.
Plant Cell 14:1737–1749
Caplan A, Berger PH, Naderi M (1998) Phenotypic variation between transgenic plants:
what is making gene expression unpredictable? In: Jain SM, Brar DS, Ahloowalia BS
(eds) Somaclonal variation and induced mutations in crop improvement. Kluwer, Dor-
drecht, pp 539–562
Chang C, Kwok SF, Bleecker AB, Meyerowitz EM (1993) Arabidopsis ethylene-response
gene ETR1: similarity of product to two-component regulators. Science 262:539–544
Chen JG, Shimomura S, Sitbon F, Sandberg G, Jones AM (2001a) The role of auxin-
binding protein 1 in the expansion of tobacco leaf cells. Plant J 28:607–617
Chen JG, Ullah H, Young JC, Sussman MR, Jones AM (2001b) ABP1 is required for
organized cell elongation and division in Arabidopsis embryogenesis. Genes Dev
15:902–911
Custers JBM, Cordewener JHG, Nöllen Y, Dons JJM, van Lookeren Campagne MM (1994)
Temperature controls both gametophytic and sporophytic development in microspore
cultures of Brassica napus. Plant Cell Rep 13:267–271
De Veylder L, Joubes J, Inze D (2003) Plant cell cycle transitions. Curr Opin Plant Biol
6:536–543
del Pozo JC, Lopez-Matas MA, Ramirez-Parra E, Gutierrez C (2005) Hormonal control of
the plant cell cycle. Physiol Plant 123:173–183
Dewitte W, Murray JA (2003) The plant cell cycle. Annu Rev Plant Biol 54:235–264
Dharmasiri S, Estelle M (2002) The role of regulated protein degradation in auxin re-
sponse. Plant Mol Biol 49:401–409
Dudits D, Bögre L, Györgyey J (1991) Molecular and cellular approaches to the analysis of
plant embryo development from somatic cells in vitro. J Cell Sci 99:475–484
Plant Hormones, Molecular Aspects 171
Dudits D, Györgyey J, Bögre L, Bakó L (1995) Molecular biology of somatic embryo-
genesis. In: Thorpe TA (ed) In Vitro embryogenesis in plants. Kluwer, Dordrecht,
pp 276–308
Eshed Y, Baum SF, Bowman JL (1999) Distinct mechanisms promote polarity establisment
in carpels of Arabidopsis. Cell 99:199–209
Fehér A, Pasternak T, Dudits D (2003) Transition of somatic plant cells to an embryogenic
state. Plant Cell Tissue Organ Cult 74:201–228
Finkelstein RR, Wang ML, Lynch TJ, Rao S, Goodman HM (1998) The Arabidopsis ab-
scisic acid response locus ABI4 encodes an APETALA 2 domain protein. Plant Cell
10:1043–1054
Francis D, Sorrell DA (2001) The interface between the cell cycle and plant growth regu-
lators: a mini review. Plant Growth Regul 33:1–12
Gallois JL, Nora FR, Mizukami Y, Sablowski R (2004) WUSCHEL induces shoot stem cell
activity and developmental plasticity in the root meristem. Genes Dev 18:375–380
Gaspar T, KeversC, Faivre-RampantO, CrèvecoeurM, Penel C, GreppinH,Dommes J (2003)
Changing concepts in plant hormone action. In Vitro Cell Dev Biol Plant 39:85–105
Gray WM, Kepinski S, Rouse D, Leyser O, Estelle M (2001) Auxin regulates SCF(TIR1)-
dependent degradation of AUX/IAA proteins. Nature 414:271–276
Gu YQ, Yang C, Thara VK, Zhou J, Martin GB (2000) Pti4 is induced by ethylene and sal-
icylic acid, and its product is phosphorylated by the Pto kinase. Plant Cell 12:771–786
Gubler F, Jacobsen JV (1992) Gibberellin-responsive elements in the promoter of a barley
high-pI alpha-amylase gene. Plant Cell 4:1435–1441
Guilfoyle T, Hagen G, Ulmasov T, Murfett J (1998) How does auxin turn on genes? Plant
Physiol 118:341–347
Hagen G, Guilfoyle T (2002) Auxin-responsive gene expression: genes, promoters and
regulatory factors. Plant Mol Biol 49:373–385
Hecht V, Vielle-Calzada JP, Hartog MV, Schmidt ED, Boutilier K, Grossniklaus U, de
Vries SC (2001) The Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASE
1 gene is expressed in developing ovules and embryos and enhances embryogenic
competence in culture. Plant Physiol 127:803–816
Hemerly AS, Ferreira P, de Almeida Engler J, Van Montagu M, Engler G, Inze D (1993)
cdc2a expression in Arabidopsis is linked with competence for cell division. Plant Cell
5:1711–1723
Henderson JT, Li HC, Rider SD, Mordhorst AP, Romero-Severson J, Cheng JC, Robey J,
Sung ZR, de Vries SC, Ogas J (2004) PICKLE acts throughout the plant to repress ex-
pression of embryonic traits and may play a role in gibberellin-dependent responses.
Plant Physiol 134:995–1005
Hirt H, Pay A, Györgyey J, Bako L, Nemeth K, Bogre L, Schweyen RJ, Heberle-Bors E,
Dudits D (1991) Complementation of a yeast cell cycle mutant by an alfalfa cDNA en-
coding a protein kinase homologous to p34cdc2. Proc Natl Acad Sci USA 88:1636–1640
Inohara N, Shimomura S, Fukui T, Futai M (1989) Auxin-binding protein located in
the endoplasmic reticulum of maize shoots: molecular cloning and complete primary
structure. Proc Natl Acad Sci USA 86:3564–3568
Inoue T, Higuchi M, Hashimoto Y, Seki M, Kobayashi M, Kato T, Tabata S, Shinozaki K,
Kakimoto T (2001) Identification of CRE1 as a cytokinin receptor from Arabidopsis.
Nature 409:1060–1063
Jiménez VM (2001) Regulation of in vitro somatic embryogenesis with emphasis on the
role of endogenous hormones. Rev Bras Fisiol Veg 13:196–223
John PCL, Zhang K, Dongg C, Diederich L, Wightman F (1993) A p34cdc2 related protein
in control of cell cycle progression, the switch between division and differentiation in
172 C. Thomas · V.M. Jiménez
tissue development and stimulation of division by auxin and cytokinin. Aust J Plant
Physiol 20:503–526
Jones AM, Im KH, Savka MA, Wu MJ, DeWitt NG, Shillito R, Binns AN (1998) Auxin-
dependent cell expansion mediated by overexpressed auxin-binding protein 1. Science
282:1114–1117
Kitamiya E, Suzuki S, Sano T, Nagata T (2000) Isolation of two genes that were induced
upon the initiation of somatic embryogenesis on carrot hypocotyls by high concentra-
tions of 2,4-D. Plant Cell Rep 19:551–557
Klucher KM, Chow H, Reiser L, Fischer RL (1996) The AINTEGUMENTA gene of Ara-
bidopsis required for ovule and female gametophyte development is related to the
floral homeotic gene APETALA2. Plant Cell 8:137–153
Kulaeva ON, Prokoptseva OS (2004) Recent advances in the study of mechanisms of
action of phytohormones. Biochemistry (Moscow) 69:233–247
Laureys F, Dewitte W, Witters E, Van Montagu M, Inze D, Van Onckelen H (1998) Zeatin
is indispensable for the G2-M transition in tobacco BY-2 cells. FEBS Lett 426:29–32
Laux T, Mayer KF, Berger J, Jurgens G (1996) The WUSCHEL gene is required for shoot
and floral meristem integrity in Arabidopsis. Development 122:87–96
Leblanc N, David K, Grosclaude J, Pradier JM, Barbier-Brygoo H, Labiau S, Perrot-
Rechenmann C (1999) A novel immunological approach establishes that the auxin-
binding protein, Nt-abp1, is an element involved in auxin signaling at the plasma
membrane. J Biol Chem 274:28314–28320
Lenhard M, Bohnert A, Jurgens G, Laux T (2001) Termination of stem cell maintenance
in Arabidopsis floral meristems by interactions between WUSCHEL and AGAMOUS.
Cell 105:805–814
Li G, Hall TC, Holmes-Davis R (2002) Plant chromatin: development and gene control.
Bioassays 24:234–243
Lin X, Hwang GJ, Zimmerman JL (1996) Isolation and characterization of a diverse set of
genes from carrot somatic embryos. Plant Physiol 112:1365–1374
Lohmann JU, Hong RL, Hobe M, Busch MA, Parcy F, Simon R, Weigel D (2001) A mo-
lecular link between stem cell regulation and floral patterning in Arabidopsis. Cell
105:793–803
Lotan T, Ohto M, Yee KM, West MA, Lo R, Kwong RW, Yamagishi K, Fischer RL, Gold-
berg RB, Harada JJ (1998) Arabidopsis LEAFY COTYLEDON1 is sufficient to induce
embryo development in vegetative cells. Cell 93:1195–1205
Luerssen H, Kirik V, Herrmann P, Misera S (1998) FUSCA3 encodes a protein with a con-
served VP1/AB13-like B3 domain which is of functional importance for the regulation
of seed maturation in Arabidopsis thaliana. Plant J 15:755–764
Marcotte WR Jr, Guiltinan MJ, Quatrano RS (1992) ABA-regulated gene expression: cis-
acting sequences and trans-acting factors. Biochem Soc Trans 20:93–97
Martinez MC, Jorgensen JE, Lawton MA, Lamb CJ, Doerner PW (1992) Spatial pattern
of cdc2 expression in relation to meristem activity and cell proliferation during plant
development. Proc Natl Acad Sci USA 89:7360–7364
Mayer KF, Schoof H, Haecker A, Lenhard M, Jurgens G, Laux T (1998) Role of WUSCHEL
in regulating stem cell fate in the Arabidopsis shoot meristem. Cell 95:805–815
McCarty DR, Carson CB, Stinard PS, Robertson DS (1989) Molecular analysis of
viviparous-1: an abscisic acid-insensitive mutant of maize. Plant Cell 1:523–532
McCarty DR, Hattori T, Carson CB, Vasil V, Lazar M, Vasil IK (1991) The viviparous-1 de-
velopmental gene of maize encodes a novel transcriptional activator. Cell 66:895–905
Meinke DW, Franzmann LH, Nickle TC, Yeung EC (1994) Leafy cotyledon mutants of
Arabidopsis. Plant Cell 6:1049–1064
Plant Hormones, Molecular Aspects 173
Meller Y, Sessa G, Eyal Y, Fluhr R (1993) DNA-protein interactions on a cis-DNA element
essential for ethylene regulation. Plant Mol Biol 23:453–463
Menke FL, Champion A, Kijne JW, Memelink J (1999) A novel jasmonate- and elicitor-
responsive element in the periwinkle secondary metabolite biosynthetic gene Str
interacts with a jasmonate- and elicitor-inducible AP2-domain transcription factor,
ORCA2. EMBO J 18:4455–4463
Miao GH, Hong Z, Verma DP (1993) Two functional soybean genes encoding p34cdc2
protein kinases are regulated by different plant developmental pathways. Proc Natl
Acad Sci USA 90:943–947
Mordhorst AP, Voerman KJ, Hartog MV, Meijer EA, van Went J, Koornneef M, de Vries SC
(1998) Somatic embryogenesis in Arabidopsis thaliana is facilitated by mutations in
genes repressing meristematic cell divisions. Genetics 149:549–563
Nagata T, Ishida S, Hasezawa S, Takahashi Y (1994) Genes involved in the dedifferentia-
tion of plant cells. Int J Dev Biol 38:321–327
Nakano Y, Steward N, Sekine M, Kusano T, Sano H (2000) A tobacco NtMET1 cDNA
encoding a DNA methyltransferase: molecular characterization and abnormal pheno-
types of transgenic tobacco plants. Plant Cell Physiol 41:448–457
Nolan KE, Irwanto RR, Rose RJ (2003) Auxin up-regulates MtSERK1 expression in both
root-forming and embryogenic cultures. Plant Physiol 133:218–230
Ogas J, Cheng JC, Sung ZR, Somerville C (1997) Cellular differentiation regulated by
gibberellin in the Arabidopsis thaliana pickle mutant. Science 277:91–94
Ogas J, Kaufmann S, Henderson J, Somerville C (1999) PICKLE is a CHD3 chromatin-
remodeling factor that regulates the transition from embryonic to vegetative develop-
ment in Arabidopsis. Proc Natl Acad Sci USA 96:13839–13844
Redig P, Shaul O, Inze D, Van Montagu M, Van Onckelen H (1996) Levels of endogenous
cytokinins, indole-3-acetic acid and abscisic acid during the cell cycle of synchronized
tobacco BY-2 cells. FEBS Lett 391:175–180
Rider SD Jr, Henderson JT, Jerome RE, Edenberg HJ, Romero-Severson J, Ogas J (2003)
Coordinate repression of regulators of embryonic identity by PICKLE during germi-
nation in Arabidopsis. Plant J 35:33–43
Rider SD Jr, Hemm MR, Hostetler HA, Li HC, Chapple C, Ogas J (2004) Metabolic profil-
ing of the Arabidopsis pkl mutant reveals selective derepression of embryonic traits.
Planta 219:489–499
Riechmann JL, Meyerowitz EM (1998) The AP2/EREBP family of plant transcription fac-
tors. Biol Chem 379:633–646
Riou-Khamlichi C, Huntley R, Jacqmard A, Murray JA (1999) Cytokinin activation of
Arabidopsis cell division through a D-type cyclin. Science 283:1541–1544
Roudier F, Fedorova E, Lebris M, Lecomte P, Györgyey J, Vaubert D, Horvath G, Abad P,
Kondorosi A, Kondorosi E (2003) The Medicago species A2-type cyclin is auxin
regulated and involved in meristem formation but dispensable for endoreduplication-
associated developmental programs. Plant Physiol 131:1091–1103
Rück A, Palme K, Venis MA, Napier R, Felle RH (1993) Patch-clamp analysis establishes
a role for an auxin binding protein in the auxin stimulation of plasma membrane
current in Zea mays protoplasts. Plant J 4:41–46
Satoh S, Kamada H, Harada H, Fujii T (1986) Auxin-controlled glycoprotein release into
the medium of embryogenic carrot cells. Plant Physiol 81:931–933
Sauter M (1997) Differential expression of a CAK (cdc2-activating kinase)-like protein
kinase, cyclins and cdc2 genes from rice during the cell cycle and in response to
gibberellin. Plant J 11:181–190
174 C. Thomas · V.M. Jiménez
Schaller GE, Bleecker AB (1995) Ethylene-binding sites generated in yeast expressing the
Arabidopsis ETR1 gene. Science 270:1809–1811
Schmidt ED, Guzzo F, Toonen MA, de Vries SC (1997) A leucine-rich repeat containing
receptor-like kinase marks somatic plant cells competent to form embryos. Develop-
ment 124:2049–2062
Schoof H, Lenhard M, Haecker A, Mayer KF, Jurgens G, Laux T (2000) The stem cell pop-
ulation of Arabidopsis shoot meristems in maintained by a regulatory loop between
the CLAVATA and WUSCHEL genes. Cell 100:635–644
Shimomura S, Watanabe S, Hiroaki I (1999) Characterization of auxin-binding protein 1
from tobacco: content, localization and auxin-binding activity. Planta 209:118–125
Somleva MN, Schmidt EDL, de Vries SC (2000) Embryogenic cells in Dactylis glomerata
L. (Poaceae) explants identified by cell tracking and by SERK expression. Plant Cell
Rep 19:718–726
Stals H, Inze D (2001) When plant cells decide to divide. Trends Plant Sci 6:359–364
Steffens B, Feckler C, Palme K, Christian M, Bottger M, Luthen H (2001) The auxin signal
for protoplast swelling is perceived by extracellular ABP1. Plant J 27:591–599
Stone SL, Kwong LW, Yee KM, Pelletier J, Lepiniec L, Fischer RL, Goldberg RB, Harada JJ
(2001) LEAFY COTYLEDON2 encodes a B3 domain transcription factor that induces
embryo development. Proc Natl Acad Sci USA 98:11806–11811
Swiatek A, Lenjou M, Van Bockstaele D, Inze D, Van Onckelen H (2002) Differential effect
of jasmonic acid and abscisic acid on cell cycle progression in tobacco BY-2 cells. Plant
Physiol 128:201–211
Takahashi Y, Kuroda H, Tanaka T, Machida Y, Takebe I, Nagata T (1989) Isolation of an
auxin-regulated gene cDNA expressed during the transition from G0 to S phase in
tobacco mesophyll protoplasts. Proc Natl Acad Sci USA 86:9279–9283
Thiel G, Blatt MR, Fricker MD, White IR, Millner P (1993) Modulation of K+ channels
in Vicia stomatal guard cells by peptide homologs to the auxin-binding protein C
terminus. Proc Natl Acad Sci USA 90:11493–11497
Thomas C, Bronner R, Molinier J, Prinsen E, van Onckelen H, Hahne G (2002) Immuno-
cytochemical localization of indole-3-acetic acid during induction of somatic embryo-
genesis in cultured sunflower embryos. Planta 215:577–583
Thomas C, Meyer D, Himber C, Steinmetz A (2004) Spatial expression of a sunflower
SERK gene during induction of somatic embryogenesis and shoot organogenesis.
Plant Physiol Biochem 42:35–42
Thomas TL (1993) Gene expression during plant embryogenesis and germination: An
overview. Plant Cell 5:1401–1410
Tiwari SB, Wang XJ, Hagen G, Guilfoyle TJ (2001) AUX/IAA proteins are active repres-
sors, and their stability and activity are modulated by auxin. Plant Cell 13:2809–2822
Ueguchi C, Sato S, Kato T, Tabata S (2001) The AHK4 gene involved in the cytokinin-
signaling pathway as a direct receptor molecule in Arabidopsis thaliana. Plant Cell
Physiol 42:751–755
Ulmasov T, Hagen G, Guilfoyle TJ (1999) Activation and repression of transcription by
auxin-response factors. Proc Natl Acad Sci USA 96:5844–5849
van der Fits L, Memelink J (2001) The jasmonate-inducible AP2/ERF-domain tran-
scription factor ORCA3 activates gene expression via interaction with a jasmonate-
responsive promoter element. Plant J 25:43–53
Wang H, Qi Q, Schorr P, Cutler AJ, Crosby WL, Fowke LC (1998) ICK1, a cyclin-dependent
protein kinase inhibitor from Arabidopsis thaliana interacts with both Cdc2a and
CycD3, and its expression is induced by abscisic acid. Plant J 15:501–510
Plant Hormones, Molecular Aspects 175
Weigel D, Alvarez J, Smyth DR, Yanofsky MF, Meyerowitz EM (1992) LEAFY controls
floral meristem identity in Arabidopsis. Cell 69:843–859
West M, Yee KM, Danao J, Zimmerman JL, Fischer RL, Goldberg RB, Harada JJ (1994)
LEAFY COTYLEDON1 is an essential regulator of late embryogenesis and cotyledon
identity in Arabidopsis. Plant Cell 6:1731–1745
Worley CK, Zenser N, Ramos J, Rouse D, Leyser O, Theologis A, Callis J (2000) Degrada-
tion of Aux/IAA proteins is essential for normal auxin signaling. Plant J 21:553–562
Yamamoto N, Kobayashi H, Togashi T, Mori Y, Kikuchi K, Kuriyama K, Tokuji Y (2005)
Formation of embryogenic cell clumps from carrot epidermal cells is suppressed by
5-azacytidine, a DNA methylation inhibitor. J Plant Physiol 162:47–54
Yasuda H, Nakajima M, Ito T, Ohwada T, Masuda H (2001) Partial characterization of
genes whose transcripts accumulate preferentially in cell clusters at the earliest stage
of carrot somatic embryogenesis. Plant Mol Biol 45:705–712
Yazawa K, Takahata K, Kamada H (2004) Isolation of the gene encoding Carrot leafy
cotyledon 1 and expression analysis during somatic and zygotic embryogenesis. Plant
Physiol Biochem 42:215–223
Yeung EC (1995) Structural and developmental patterns in somatic embryogenesis. In:
Thorpe TA (ed) In vitro Embryogenesis in Plants. Kluwer Academic Publishers, Dord-
recht, p 205–248
Zhang K, Letham DS, John PC (1996) Cytokinin controls the cell cycle at mitosis by
stimulating the tyrosine dephosphorylation and activation of p34cdc2-like H1 histone
kinase. Planta 200:2–12
Zhao J, Morozova N, Williams L, Libs L, Avivi Y, Grafi G (2001) Two phases of chromatin
decondensation during dedifferentiation of plant cells: distinction between compe-
tence for cell fate switch and a commitment for S phase. J Biol Chem 276:22772–22778
Zimmerman JL, Thomine S, Guern J, Barbier-Brygoo H (1994) An anion current at the
plasma
Zuo J, Niu QW, Frugis G, Chua NH (2002) The WUSCHEL gene promotes vegetative-to-
embryonic transition in Arabidopsis. Plant J 30:349–359