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Shoot organogenesis, one of the in vitro plant regeneration processes that occur during in vitro micropropagation, is used in the study of plant development. Morphological, physiological, and molecular aspects of in vitro shoot organogenesis have been extensively studied for over 50 years. Because of the research progress in plant genetics and molecular biology, our understanding of in planta and in vitro shoot meristem development, the cell cycle and cytokinin signal transduction has advanced significantly. These research advances provide useful information as well as molecular tools to study further the genetic and molecular aspects of shoot organogenesis. A number of key molecular markers, genes, and pathways have been shown to play a critical role in the process of in vitro shoot organogenesis. Furthermore, these studies reveal that in vitro shoot organogenesis, as with in planta shoot development, is a complex, well-coordinated developmental process, given that the induction of a single molecular event is likely to be insufficient to induce the entire process. Continued study is required to identify additional molecular events that trigger dedifferentiation and act as developmental switches for de novo shoot development. *This article is dedicated to the memory of Shibo Zhang, who continues to inspire our interests in this topic.
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Critical Reviews in Plant Sciences
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Toward Molecular Understanding of In Vitro and In
Planta Shoot Organogenesis
Ling Meng a , Shibo Zhang a & Peggy G. Lemaux a
a Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA
Published online: 09 Mar 2010.
To cite this article: Ling Meng , Shibo Zhang & Peggy G. Lemaux (2010) Toward Molecular Understanding of In Vitro and In
Planta Shoot Organogenesis, Critical Reviews in Plant Sciences, 29:2, 108-122, DOI: 10.1080/07352681003617327
To link to this article: http://dx.doi.org/10.1080/07352681003617327
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Critical Reviews in Plant Sciences, 29:108–122, 2010
Copyright © Taylor & Francis Group, LLC
ISSN: 0735-2689 print / 1549-7836 online
DOI: 10.1080/07352681003617327
Toward Molecular Understanding of
In Vitro
and
In Planta
Shoot Organogenesis
Ling Meng, Shibo Zhang,and Peggy G. Lemaux
Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA
Table of Contents
I. INTRODUCTION .............................................................................................................................................109
II. MOLECULAR ANALYSIS OF IN PLANTA SHOOT MERISTEM DEVELOPMENT ......................................109
A. Genetic Control of SAM Patterning and Development ..................................................................................... 110
1. KNOX I and Asymmetric Leaves 1 and 2 Regulate Shoot Meristem Development ..................................... 110
2. Second Pathway Regulating SAM Activity ............................................................................................ . 111
3. Regulators of SAM Activity Via Interaction With WUS Signaling Pathway ...............................................112
B. Phytohormones Regulate SAM Patterning and Development ............................................................................ 112
1. High CK Activity Promotes Meristem Function ...................................................................................... 112
2. High Auxin and Gibberellic Acid Activities Facilitate Lateral Organogenesis ............................................ 113
C. Cross-Talk between KNOX I/WUS and the Phytohormone Pathway Specifies Spatial Cues for Cell Identity in the
SAM ...........................................................................................................................................................114
1. KNOX I Is a Major Activator of Meristematic Activity ............................................................................ 114
2. WUS Regulates CK Signaling Response ............................................................................................... . 114
3. Negative Feedback Loop between Auxin Signals and CUC and KNOXI in the SAM ................................. 114
III. MOLECULAR ANALYSIS OF PLANT CELL DIVISION ............................................................................... 115
A. Cyclin-Dependent Kinase Complexes ............................................................................................................ 115
1. Cyclin-Dependent Kinases (CDKs) ........................................................................................................ 115
2. Cyclins ................................................................................................................................................ 116
B. Regulation of Cell Cycle during Plant Development ........................................................................................ 116
1. Genetic Factors Regulate Plant Cell Cycle and Meristem Activity ............................................................ 116
2. Exogenous Signals Regulate the Plant Cell Division Cycle ....................................................................... 116
IV. MOLECULAR ANALYSIS OF IN VITRO SHOOT ORGANOGENESIS ..........................................................117
A. Use of Specific Genes as Molecular Markers .................................................................................................. 117
B. Identification of Genes Involved in In Vitro Shoot Organogenesis .. ..... .... ..... ..... .... ..... .... ..... ..... .... ..... .... ..... ..... .. 118
V. SUMMARY ......................................................................................................................................................118
ACKNOWLEDGMENTS ........................................................................................................................................... 119
REFERENCES ..........................................................................................................................................................119
Address correspondence to Peggy G. Lemaux, Department of Plant
and Microbial Biology, University of California, Berkeley, CA 94720.
E-mail: lemauxpg@berkeley.edu
This article is dedicated to the memory of Shibo Zhang, who
continues to inspire our interests in this topic.
Shoot organogenesis, one of the in vitro plant regeneration pro-
cesses that occur during in vitro micropropagation, is used in
the study of plant development. Morphological, physiological, and
molecular aspects of in vitro shoot organogenesis have been exten-
sively studied for over 50 years. Because of the research progress
in plant genetics and molecular biology, our understanding of in
108
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PLANT SHOOT ORGANOGENESIS 109
planta and in vitro shoot meristem development, the cell cycle and
cytokinin signal transduction has advanced significantly. These re-
search advances provide useful information as well as molecular
tools to study further the genetic and molecular aspects of shoot
organogenesis. A number of key molecular markers, genes, and
pathways have been shown to play a critical role in the process of
in vitro shoot organogenesis. Furthermore, these studies reveal that
in vitro shoot organogenesis, as with in planta shoot development, is
a complex, well-coordinated developmental process, given that the
induction of a single molecular event is likely to be insufficient to
induce the entire process. Continued study is required to identify
additional molecular events that trigger dedifferentiation and act
as developmental switches for de novo shoot development.
Keywords auxin, cell cycle, cytokinin, dedifferentiation, develop-
ment, in vitro organogenesis, shoot apical meristem
I. INTRODUCTION
Both somatic embryogenesis and shoot organogenesis can be
involved in the process of in vitro culturing of plant tissue. Dur-
ing shoot organogenesis an adventitious shoot forms, followed
by the development of adventitious roots from the shoot, result-
ing in an entire plant. The process of in vitro shoot induction and
development via organogenesis has been reviewed in detail from
the perspective of developmental biology (Hick, 1994) and from
a physiological, biochemical, and molecular viewpoint (Zhang
et al., 2004b). In this review, we will update a previous review
(Zhang et al., 2004a) with more recent molecular details of in
vitro and in planta shoot organogenesis.
In planta shoot development from an embryo differs from the
process of in vitro shoot organogenesis since in the latter case
the shoot meristems initiate from differentiated somatic cells,
not from embryonic cells as it does during embryogenesis. The
process of in vitro shoot organogenesis usually involves three
main stages: response of somatic cells to exogenous hormones,
cell division of responding cells, and initiation and development
of new shoots either directly from the newly dividing cells or
indirectly through a callus phase. When somatic cells respond
appropriately to exogenously applied plant hormones, they can
activate or accelerate the timing of the cell cycle, resulting in
reprogramming of cells that assume the de novo developmental
fate of shoot organogenesis.
Exogenously applied cytokinins (CKs), sometimes in con-
cert with auxins, are the most efficient plant growth regulators
that induce in vitro shoot organogenesis. Genetic and molecular
analyses led to the identification in Arabidopsis thaliana
(Arabidopsis) of important genes involved in the CK signal
transduction pathway (Haberer et al., 2002). The Arabidopsis
histidine protein kinases (AHKs) serve as CK receptors and the
histidine phosphotransfer proteins (AHPs) transmit the signal
from AHKs to nuclear response regulators (ARRs), which
activate or repress transcription. A few key regulatory genes
were shown to be involved in the plant cell cycle, such as
p34cdc2 and cyclin genes (Shaul, 1996). During shoot meristem
development, several other regulatory genes were identified,
e.g., maize KNOTTED1 (KN1) (Vollbrecht,etal., 1991), Ara-
bidopsis SHOOT MERISTEMLESS (STM) (Long et al., 1996),
WUSCHEL (WUS) (Laux et al., 1996, Mayer et al., 1998),
and CLAVATA 1-3 (CLV1-3) (Fletcher, 1999). Studies suggest
that molecular interactions occur among three processes: CK
reception, cell cycle, and shoot meristem development (Riou-
Khamlichi et al., 1999). Identification of these genes suggests
that they could be used as molecular markers to develop a better
understanding of in vitro shoot organogenesis. Such approaches
have also been used to identify genes more directly involved
with in vitro shoot organogenesis. The current molecular under-
standing of d in vitro shoot meristem development, cell cycle
regulation, and CK signal transduction will be described in this
review.
II. MOLECULAR ANALYSIS OF
IN PLANTA
SHOOT
MERISTEM DEVELOPMENT
During zygotic embryogenesis the shoot meristem initiates
and develops within the embryo that derives from the zygote.
Because of its position at the growing tip, the initial shoot meris-
tem becomes the shoot apical meristem (SAM), which gener-
ates all stems, leaves, and lateral shoot meristems during the
entire process of shoot development (Kwiatkowska, 2008). The
SAM, which is located at the shoot apex above the youngest
leaf primordium, provides cells for new organ initiation from
a pool of cells that undergoes continuous renewal by cell
division.
Based on histological and anatomical analyses, the SAM can
be subdivided into three distinct radial layers (L1, L2 and L3,
Figure 1(b)) and zones [peripheral (PZ), central (CZ) and rib
(RZ), Figure 1(a)]. L1 and L2 cells form the tunica, dividing
exclusively in an anticlinal manner to maintain the tunica layer.
L3 cells, constituting the corpus tissue, divide both anticlinally
and periclinally. The L1 layer gives rise to the epidermis, L2 to
mesophyll cells that provide germ line cells that form gametes,
and L3 cells to central tissues of the leaf and stem. Lateral or-
gans originate from the PZ; stem tissue derives from the RZ.
The CZ holds the stem cell pool that continuously replenishes
itself and cells in the PZ and RZ (Kwiatkowska, 2008). The size
and the number of stem cells in the SAM appear generally con-
stant, suggesting that the continuous process of cell division and
differentiation of daughter cells into organ primordia appears to
be well-balanced.
Plant shoot development from the SAM depends on cor-
rect cell-fate determination and maintenance of a proper stem
cell pool and specification of organ (leaf) founder cells from
the PZ where lateral organs arise. Cell fate is thought to be
largely determined by the spatial position of cues (pattern for-
mation) within a morphogenetic field during critical time pe-
riods not by cell lineage via inherited cues or gene expres-
sion (Scheres, 2001). Spatial specification of organ-founder and
stem cells is thus critical to proper shoot meristem activity and
development.
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110 L. MENG ET AL.
FIG. 1. Diagrams of the shoot apical meristem (SAM) (a) showing the central zone (CZ), peripheral zone (PZ), and rib zone (RZ) and (b) showing the L1, L2,
and L3 layers.
A. Genetic Control of SAM Patterning and Development
Intercellular communication is critical in controlling SAM
organization and maintenance, as well as cell-fate specification
(Williams et al., 2005). Genetic and molecular analyses of shoot
meristem development in maize, Arabidopsis and other plants
have led to identification of genes critical to this process.
1. KNOX I and Asymmetric Leaves 1 and 2 Regulate Shoot
Meristem Development
The gene for the maize homeodomain protein, Knotted
1(KN1) which causes “knots” when misexpressed in leaves
(Vollbrecht et al., 1991), is expressed exclusively in the SAM
and is required for its activity and development (Jackson et al.,
1994). Expression patterns of KN1-homologs in other cereals,
like barley, are consistent with this pattern (Figure 2). The KNOX
(Knotted-like homeobox) gene family, found in all plant species,
is divided into two classes based on expression patterns. Class
IKNOX (KNOX I) genes are specifically expressed in the SAM
and promote meristem activity, whereas class II KNOX genes are
more widely expressed in plant tissues (Kerstetter et al., 1994).
Relevant to shoot organogenesis, ectopic expression of maize
KN1 in tobacco, or its orthologs in Arabidopsis, consistently
causes adventitious shoot formation in leaf tissues (Lincoln
et al., 1994; Sinha et al., 1993). Misexpression of KN1 in the bar-
ley awn induces ectopic meristems that form inflorescence-like
structures on the awn (Williams-Carrier et al., 1997) a phe-
notype similar to that in the dominant gain-of-function hooded
mutant that is caused by misexpression of HvKNOX3.
Four KNOX I protein family members were found in Ara-
bidopsis: SHOOT MERISTEMLESS (STM), BREVIPEDI-
CELLUS (BP), KN1-like in Arabidopsis thaliana 2(KNAT2)
and KNAT6. STM, the first KNOX gene shown to have recessive
loss-of-function phenotypes, is most similar to maize KN1 in
terms of expression pattern and function (Long et al., 1996).
FIG. 2. Expression of the KNOTTED1-homolog in a shoot apical meristem (SAM) of barley. (a) Scanning electron micrograph of a barley shoot apex including
the SAM and two leaf primordia (P1, P2). (b) Immunolocalization of KNOTTED1-homolog in barley shoot apex using KNOTTED1 antibody, showing expression
of KNOTTED1-homolog only in the SAM, not in leaf primordia (P0, P1, and P2).). (From Trigiano, R. N. and Gary, D. J., Eds., Plant Tissue Culture and
Development, CRC Press LLC, Boca Raton, FL, 2004).
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PLANT SHOOT ORGANOGENESIS 111
STM expression, required to maintain all shoot meristems, is
restricted to the SAM, starting in the embryo in a single api-
cal cell at the late globular stage (Long et al., 1998). Loss-of-
function mutations of STM result in premature termination of the
SAM (Long et al., 1996). STM and BP act together to maintain
SAM activity by preventing meristematic cells from developing
organ (leaf)-specific cell fates (Byrne et al., 2002). KNAT2 and
KNAT6, which share higher sequence similarity to one another
than to STM or BP, do not share identical functions; KNAT6,
but not KNAT2, regulates SAM activity and organ separation
in the embryo. Mutations in KNAT2 and KNAT6 result in an-
tagonistic interactions with BP; expression of both increases in
bp mutants, suggesting BP represses expression of KNAT2 and
KNAT6 (Ragni et al., 2008).
Correct cell fate determination in the SAM depends on mu-
tual expression/repression between Asymmetric Leaves 1 (AS1)
and AS2 and KNOX I genes. The latter, which promote meris-
tem activity, are expressed in the SAM but are downregulated in
founder cells at leaf initiation (Jackson et al., 1994). AS1 encodes
a myb domain transcription factor closely related to PHANTAS-
TICA (PHAN)inAntirrhinum and ROUGH SHEATH2 (RS2)in
maize. All three genes, which are expressed in leaf founder cells,
influence leaf fate. AS1 represses KNAT1 and KNAT2 expression
and is itself negatively regulated by STM. In loss-of-function
as1 mutants occasional lobes appear on the leaves similar to
those in 35S:BP plants (Byrne et al., 2000, Ori et al., 2000). BP,
KNAT2, and KNAT6, but not STM, are ectopically expressed in
leaves of as1 mutants. AS1 expression expands into the SAM
in stm mutants, consistent with its restricting AS1 expression.
AS2, which encodes a LATERAL ORGAN BOUNDARIES
(LOB) domain transcription factor, is expressed mainly in adax-
ial domains of leaf primordia. Phenotypes of as2 mutants are
similartothoseofas1, with rumpled, slightly lobed leaves. AS2
is believed to control formation of symmetric flat leaf lamina
and establish a prominent midvein and other patterns of venation
(Iwakawa et al., 2002); it also appears to restrict KNOX I expres-
sion. Expression from BP,KNAT2, and KNAT6, but not STM,
is ectopic in as2 mutants. AS1, acting with AS2, directly binds
BP and KNAT2 promoters and may form a repressive chromatin
complex (Guo et al., 2008). This mutual interaction between
AS1,AS2 and KNOX I defines a mechanism for differentiating
stem and organ founder cells within the SAM, demonstrating
that genes expressed in organ primordia interact with meristem-
atic genes to regulate shoot morphogenesis (Byrne et al., 2000;
Byrne et al., 2002). BLADE-ON-PETIOLE1 (BOP1) encodes a
transcription factor involved in regulating leaf differentiation;
BOP1 is thought to down-regulate KNOX I expression dur-
ing leaf formation. KNAT1, KNAT2 and KNAT6 are expressed
ectopically in leaves of the bop1-1 mutant. BOP1 has synergis-
tic effects with ASI or AS2 in bop1/as1 or bop1/as2 double
mutants, and with STM in bop1/stm-1 double mutants suggest-
ing it promotes leaf development via KNOX1 repression (Ha,
2004).
2. Second Pathway Regulating SAM Activity
Local signaling to maintain stem cells in the SAM in Ara-
bidopsis involves the CLAVATA-WUSCHEL (CLV-WUS) sig-
nal transduction pathway. WUS, distantly related to STM home-
odomain transcription factors, acts non-cell autonomously to
promote stem cell fate. It is first expressed at the 16-cell stage in
subepidermal cells of the SAM but ultimately becomes limited
to a few central cells of the RZ in the organizing center (OC)
(Mayer et al., 1998). In wus mutants, shoot meristem develop-
ment stops after the first few leaves, resulting in bushy plants
(Laux et al., 1996). Ectopic WUS expression in roots induces
shoot meristems and organs, such as leaves (Gallois et al., 2004).
Thus, WUS can establish stem cells with shoot meristem iden-
tity and can maintain stem cells in the CZ of the SAM. WUS
and STM are activated independently in Arabidopsis; however,
expression from STM is absent in wus mutants and vice versa
(Lenhard, 2002).
WUS induces expression from CLAVATA3 (CLV3), which
encodes a small extracellular signal ligand (Fletcher, 1999) that
is present in stem cells of the CZ. CLV3 is secreted from L1
and L2 toward interior stem cells of the CZ to directly interact
(Ogawa et al., 2008) with CLAVATA 1 (CLV1), a leucine-rich-
repeat (LRR) receptor kinase (Clark et al., 1997) that forms a
heterodimeric complex with CLAVATA 2 (CLV2) to suppress
WUS expression. This feedback regulation allows the SAM to
balance stem cell division in the CZ with cell differentiation in
the PZ.
CLV3 is expressed in L1 and L2 layers, CLV1 onlyinL3ofthe
CZ (Clark et al., 1997), whereas CLV2 transcripts are detected in
most plant tissues (Jeong et al., 1999). Loss-of-function CLV1,
CLV2 or CLV3 mutations result in expanded WUS expression,
enlarged shoot and floral meristems, and flowers that contain
extra organs (Clark et al., 1997). Conversely, overexpression of
CLV3 causes loss of WUS expression and premature shoot and
floral meristem termination, showing that interaction between
secreted CLV3 and CLV1/CLV2 receptor complex limits the
size of the WUS expression domain and thus the meristem.
The CLV3/WUS signaling pathway forms a spatial, negative
feedback loop that controls stem cell accumulation in shoot
and floral meristems (Figure 3) [for review, see Williams et al.,
(2005)].
More than 40 CLV3/ESR (EMBRYO SURROUNDING
REGION) -related (CLE) proteins have been identified in plants
and plant-parasitic nematodes. The CLE family encodes small,
putative signal ligands with a conserved 14-amino acid motif
at the C-terminus and a signal peptide at the N-terminus (Cock
et al., 2001). A predominant ectopic overexpression phenotype
for some CLE genes is premature SAM termination (Strabala
et al., 2006). Of note, a 12-amino-acid modified peptide derived
from the CLE motif was observed in transgenic Arabidopsis
callus overexpressing CLV3 (Kondo et al., 2006). When used
in culture media, the synthetic 12- or 14-amino acid peptides
from several CLE motifs phenocopied their gain-of-function
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112 L. MENG ET AL.
phenotypes, and in some cases rescued clv3 loss-of-function
phenotypes (Fiers et al., 2005). Together, these results suggest
that CLE negatively regulates meristem activity; however, there
is no direct evidence for CLV3 peptide-ligand interaction with
the CLV1/CLV2 receptor complex (Ogawa et al., 2008).
3. Regulators of SAM Activity Via Interaction With WUS Sig-
naling Pathway
Besides CLV genes, several signaling components, which
regulate SAM via interaction with the WUS pathway, were iden-
tified. ULTRAPETALA1 (ULT1), a DNA binding-domain tran-
scription factor found in all meristems and developing stamens,
carpels and ovules, restricts SAM size by repressing WUS ex-
pression. ULT1 appears to have WUS-independent functions in
maintaining SAM activity given that an ult1/wus double mutant
shows additive phenotypes (Williams et al., 2005). HANABA
TARANU (HAN), a GATA-3-like transcription factor, is also a
WUS signaling pathway component. HAN, transcribed at the
boundaries between the meristem and its newly initiated organ
primordia and in vascular tissues, establishes organ boundaries
in shoots and controls the number and correct positioning of
WUS-expressing cells.
BARD1 (BRCA1-associated RING domain 1) protein, con-
taining a RING domain and two tandem BRCA1 C-terminal
domains that function in phosphorylation-dependent protein–
protein interactions, interacts directly with the WUS promoter
region. A bard1 mutant has severe SAM defects, increased WUS
expression and overexpression of BARD1 which causes a wus
mutant phenotype. BARD1 is thought to be responsible for lim-
iting WUS expression and regulating SAM organization and
maintenance (Han et al., 2008).
Two widely expressed homeodomain finger proteins
OBERON1 (OBE1) and OBE2 function redundantly to
allow plant cells to acquire meristematic activity via the
WUSCHEL-CLAVATA pathway. In obe1/obe2 double mutants,
the shoot meristem prematurely terminates with a dramatic
reduction in CLV3 and WUS expression. OBE1 and OBE2 are
believed to be responsible for cells reaching a state that leads
to establishment and maintenance of the meristem (Saiga et
al., 2008). Class III homeodomain-leucine zipper (HD-ZIP III)
transcription factors also appear involved in SAM maintenance
by modulating WUS transcription levels in the OC. Arabidopsis
contains five HDZIP III genes, CORONA/ATHB15 (CNA),
REVOLUTA/INTERFASCICULAR FIBERLESS1 (REV),
PHABULOSA (PHB), PHAVOLUTA (PHV) and ATHB8.
Although cna single mutants exhibit subtle defects in meristem
development, clv/cna double mutants develop a massively
enlarged and disorganized SAM and ectopic expression of
WUS and CLV3. Thus, CNA is believed to regulate stem
cell identity in a pathway parallel to that of CLVs (Williams
et al., 2005). The triple mutant, can/phb/ phv, recreates the
clv enlarged-SAM phenotype. Thus the CNAs appear to
regulate SAM patterning and meristem function in a complex
pathway.
Expression from WUS is regulated epigenetically by chro-
matin remodeling. Loss-of –function mutants in FA S C I A TA 1
(FA S 1 )orFA S 2 , encoding two subunits of the chromatin as-
sembly factor 1 complex that function by regulating WUS ex-
pression, have a flat, enlarged, disorganized SAM (Williams
et al., 2005). In fas mutants, the WUS expression domain ex-
pands laterally, but not uniformly, resulting in a varied pattern.
WUS may also be a direct target of the chromatin remodeling
factor, SPLAYED (SYD), which interacts with the WUS pro-
moter, giving evidence that WUS expression is regulated at the
chromatin level. Mutants in syd with reduced transcription levels
from WUS and CLV3 prematurely terminate the SAM.
Besides the KNOX I-AS1 and WUS-CLV pathways,
other genes are involved in shoot meristem development
and maintenance. CUP-SHAPED COTYLEDON1 (CUC1)
and CUC2 are essential for meristem establishment and in
forming boundaries between meristems and adjacent organs.
Both CUC1 and CUC2, which are expressed in a strip of
cells at the apex of the globular-stage embryo, are involved
in embryonic SAM formation and cotyledon separation (Aida
et al., 1997; Takada et al., 2001). Seedlings of the cuc1/
cuc2 double mutant lack an embryonic SAM, and the two
cotyledons are fused along both edges to form a cup-shaped
structure (Aida et al., 1997). CUC1 and CUC2, thought to
function upstream of STM, regulate SAM formation through
transcriptional activation of STM [for review, see Fletcher et al.
(2000)].
In summary, in planta and in vitro shoot meristem devel-
opment and maintenance involve a large number of genes that
orchestrate a complicated and well-regulated process. Certain
aspects of the molecular mechanisms involved have been dis-
covered; however, full elucidation of all of the pathways still
requires additional research.
B. Phytohormones Regulate SAM Patterning and
Development
In addition to genetic factors, phytohormones, e.g., auxins
and CKs, play major roles in controlling plant development.
During in vitro culture an excess of CK over auxin promotes
shoot formation in callus, while auxin excess induces root for-
mation (Skoog et al., 1957). Evidence suggests that differen-
tially distributed hormone activities across the SAM seem to be
linked to basic aspects of meristem activity and development
(Veit, 2009). Relatively high levels of auxin and gibberellic acid
(GA) are closely associated with initiation and outgrowth of lat-
eral organs in the PZ, whereas, high levels of CK in the CZ can
be linked to maintenance of a reservoir of undetermined cells
that enable indeterminate growth.
1. High CK Activity Promotes Meristem Function
CKs play crucial roles in plant development, i.e., promot-
ing cell division, shoot meristem initiation, root and vascula-
ture patterning, chloroplast biogenesis and photomorphogene-
sis. Besides inducing shoot meristems from callus during in vitro
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PLANT SHOOT ORGANOGENESIS 113
culture, high levels of CK relative to auxin in the shoot apex is re-
quired for SAM function. This was first noted in the Arabidopsis
amp1 (altered meristem program1) mutants, which have higher
than normal CK levels, an enlarged SAM and increased cell
proliferation and cyclin CYCD3 expression (Helliwell et al.,
2001). Another clue to the role of CK in shoot meristem de-
velopment is the rice gene, Lonely Guy (LOG), which encodes
a novel CK-activating enzyme, the final step in bioactive CK
synthesis; loss-of-function LOG causes premature termination
of the shoot meristem. LOG is expressed specifically in the
shoot apex, suggesting specific activation of CKs as a mecha-
nism to regulate SAM function (Kurakawa et al., 2007). Other
studies suggest that decreasing CK by overexpressing CK ox-
idases reduces meristem size and sometimes causes meristem
termination (Werner et al., 2003).
CK response involves multiple steps involving two-
component circuitry through a histidine and aspartate phos-
phorelay signaling pathway. Two-component AHKs contain a
variable CK sensor and a conserved histidine kinase domain
that acts as a CK receptor. AHPs act as signaling shuttles be-
tween CK receptors and downstream nuclear responses and are
translocated to the nucleus upon phosphorylation by an activated
AHK, thus relaying the phosphate to ARRs, which regulate a
transcriptional network that controls plant responses (Kakimoto,
2003).
Using activation-tagging that causes overexpression of a
tagged gene for a putative CK receptor, cytokinin-independent1
(CKI1) was identified [for review, see Kakimoto (2003)]. CKI1
encodes a protein similar to two-component regulators, having
histidine kinase and receiver domains, thus resembling a CK
receptor. Overexpression of CKI1 results in a response indepen-
dent of CK elevated cell division and greening suggesting
that CKI1 is involved in CK signaling, perhaps as a CK re-
ceptor. Subsequently CYTOKININ RESPONSE 1 (CRE1), also
known as AHK4 or WOL (WOODEN LOG) (Ueguchi et al.,
2001), was identified (Inoue et al., 2001) and confirmed as en-
coding a receptor that binds CK directly (Yamada et al., 2001).
Cells from a cre1 mutant displayed reduced sensitivity to CK
in vitro. Analyses in yeast and E. coli indicate that CK can act
as a ligand for CRE1/AHK4 (Inoue et al., 2001; Yamada et al.,
2001). CRE1/AHK4 and two homologues, AHK2 and AHK3,
share a CK-binding domain with varying numbers of transmem-
brane segments. In protoplasts transient overexpression of any
of the receptors increased CK sensitivity (Hwang et al., 2001),
suggesting partially redundant functions (Higuchi et al., 2004;
Riefler et al., 2006).
Six AHPs have been identified in Arabidopsis. AHP1 and
AHP2 translocate to the nucleus after CK treatment (Hwang
et al., 2001). Loss-of-function mutation analysis revealed strong
functional redundancy of AHPs; single ahp mutants were in-
distinguishable in CK response from wild-type, while higher
order mutants displayed variable reduced sensitivity to CK. A
quintuple ahp1, 2, 3, 4, 5 mutant showed the most apparent
reduction in CK sensitivity (Hutchison, et al., 2006). AHP6,
a pseudophosphotransfer protein lacking the conserved His re-
quired for phosphotransfer, negatively regulates CK, likely by
competing with functional AHPs for AHK interaction. Alterna-
tively, the presence of AHP6 in a specific spatial domain, may
limit CK activity, contributing to fine-tuning cell differentiation
boundaries (M¨
ah¨
onen et al., 2006).
ARRs, which share a receiver domain with conserved Asp
residues, are classified based on structure and CK-inducible ex-
pression patterns into two subtypes, A and B (Imamura et al.,
1999). Upon activation, nuclear B-types function as transcrip-
tional factors, activating target gene transcription, including A-
types (Hwang et al., 2001). Conversely, CK-inducible A-types
repress CK signaling, and thus may act as negative regulators
of CK response in a negative-feedback loop. These studies sug-
gest a multistep phosphorelay pathway is involved in CK signal
transduction: CRE1/AHK4/WOL AHPs ARRs re-
sponse.
A close correlation exists between CK response and SAM ac-
tivity. Some type A ARRs, e.g., ARR7, negatively regulate SAM
activity. Overexpression of ARR7 causes reduced meristem ac-
tivities, resulting in wus-like phenotypes (Leibfried et al., 2005).
Similarly, loss-of-function mutants of the maize phyllotaxis-
controlling gene, ABPH1 encoding a CK-inducible type A ARR,
have increased meristem size and altered phyllotaxy patterns.
Since ABPH1 is expressed specifically in the youngest leaf
primordia, it was proposed that it function in limiting SAM
size through negative regulation of CK signaling (Giulini et
al., 2004). Moreover, loss-of-function of the three CK recep-
tors, CRE/AHK4/WOL, AHK2 AHK3, resulted in reduction in
shoot meristem size and cell proliferation (Higuchi et al., 2004).
Based on the results from all of these studies, it seems that
higher CK activity relative to auxin in the CZ is required for
optimal SAM function.
2. High Auxin and Gibberellic Acid Activities Facilitate Lat-
eral Organogenesis
Auxin, being central to many aspects of plant development,
is produced primarily in young leaves and primordia and
is transported to sites where it functions. It controls plant
development through gradients and maxima established by
auxin efflux carriers, i.e., the PIN (PINFORMED) family that
drives polar auxin transport that maximizes in leaf primordia
on the flank of the SAM, thus aiding lateral organ formation
(G¨
alweiler et al., 1998). Loss-of-function mutations of PIN1, or
chemical treatment with an auxin transport inhibitor that causes
loss of auxin maxima, blocks lateral organ formation (Benkova
et al., 2003; Scanlon, 2003). The block can be overcome
by exogenous auxin, which restores leaf formation at the
application site (Reinhardt et al., 2000; Reinhardt et al., 2003).
Altering polar distribution by exogenous auxin application
to Brassica juncea embryos resulted in cotyledon fusion and
failure of further organ formation (Hadfi et al., 1998).
SAMs have a gradient of auxin concentration with high
levels at the PZ, decreasing toward the CZ. Differential auxin
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114 L. MENG ET AL.
distribution is regulated by the influence of CK on cell-to-cell
polar auxin transport by altering expression of several PIN
proteins (Pernisov´
aet al., 2009). Another possibility is that the
auxin rapidly decreases CK biosynthesis in the SAM, which
may locally affect the CK gradient from the CZ to the PZ (Nord-
strom et al., 2004). High auxin to CK ratios, not absolute auxin
concentrations, might trigger lateral organ initiation since their
primordia initiate only from the PZ, not the CZ, even though
auxin is present in the CZ (Treml et al., 2005). Since exogenous
auxin application at the CZ does not induce lateral organ initia-
tion (Reinhardt et al., 2003), additional factors appear to operate.
Specific binding of Aux/IAA proteins to the transcription fac-
tor, ARF (auxin response factor), prevents ARFs from activating
transcription of auxin-inducible genes, including Aux/IAA. This
binding targets proteins for degradation in an auxin-dependent
manner through ubiquitin-mediated protein degradation, result-
ing in activation of an auxin response (Gray et al., 2001).
Dominant negative, loss-of-function, mutant alleles of the co-
repressor, TOPLESS (PTL), cause the embryonic SAM to be-
come root-like, suggesting PTL represses root development in
apical regions of the developing embryo (Long et al., 2006). De-
fects may also result from limiting ARF5/MP activities in the
CZ, not by altering auxin distribution (Szemenyei et al., 2008).
Gibberellic acid (GA) also regulates many processes in
plant development, including leaf initiation and outgrowth and
morphogenesis (Fleet et al., 2005). Similar to auxin, GA levels
are high in leaf primordia and young leaves, but low in the SAM
(Hay et al., 2004). Tight spatial control of GA accumulation ap-
pears crucial for establishing boundaries between meristematic
and leaf founder cells (Sakamoto et al., 2001); repression of
GA in the SAM maintains meristem function (Hay et al., 2004).
Synthesis and degradation of GA are both controlled by KNOX
I and auxin; auxin promotes GA synthesis in decapitated stems
(Ross et al., 2000; Wolbang et al., 2001), while KNOX I
is required to maintain low GA levels in the SAM (Jasinski
et al., 2005).
Expression from KNOX I in the SAM excludes expression of
the GA biosynthetic gene, GA20ox, the rate-limiting step in GA
biosynthesis (Hay et al., 2004). In tobacco and potato, KNOX
Igenes bind to the GA20ox promoter, restricting its expression
in the meristem periphery (Chen et al., 2004; Sakamoto et al.,
2001). KNOX I genes further restrict GA synthesis by promoting,
in a ring surrounding the SAM, transcription of GA2ox2, which
mediates GA inactivation (Jasinski et al., 2005; Sakamoto et al.,
2001). Maize KN1 directly regulates expression from ga2ox1
by binding to an intron (Bolduc et al., 2009). No such restric-
tion of GA occurs in lateral organ primordia, where KNOX I
genes are partially repressed by high auxin activities (Hay et al.,
2006). Expression from ga2ox2 is likely regulated by other fac-
tors since expression only partially overlaps with that of STM.
A mutant of SPINDLY (SPY), another negative regulator of
GA, is insensitive to exogenous CK application but has a con-
stitutive GA signaling response (Greenboim-Wainberg et al.,
2005).
C. Cross-Talk between KNOX I/WUS and the
Phytohormone Pathway Specifies Spatial Cues for
Cell Identity in the SAM
Study of mechanisms underlying the role of KNOX and
WUS pathways and phytohormone activities in maintaining
SAM function show that KNOX I and WUS directly link to
phytohormone pathways. This results from a local CK activ-
ity gradient high auxin at the CZ decreasing towards the PZ.
High auxin relative to CK and GA represses KNOX I and CUC
expression and CK activity, resulting in specification of lateral
organ primordia.
1. KNOX I Is a Major Activator of Meristematic Activity
Direct molecular evidence for positive regulation of CK
biosynthesis by KNOX1s is that misexpression of STM results
in a rapid increase in expression from ISOPENTENYLTRANS-
FERASE7 (IPT7), which encodes an enzyme involved in CK
biosynthesis and accumulation. A severe mutant of the CK re-
ceptor, AHK4/CRE1/WOL, resulted in a weak stm allele (Jasin-
ski et al., 2005); exogenous application of CK or expression of
bacterial IPT in the SAM partially restores meristem activity
(Yanai et al., 2005). Thus, STM promotes SAM activity at least
partially by activating CK biosynthesis in the SAM, but high
CK activity also results in increased KNAT1 and STM transcript
levels (Rupp et al., 1999).
2. WUS Regulates CK Signaling Response
A direct link was identified between WUS and CK signaling
that induces stem cell identity. WUS directly represses expres-
sion of several CK-inducible type A ARRs, which act in a
negative feedback loop of CK signaling in the SAM (Leibfried
et al., 2005). ARR7 expression is limited specifically to the PZ
and WUS was shown to bind upstream of the ARR7 transcrip-
tion start site. Ectopic activation of ARR7 causes a wus loss-
of-function phenotype. A mutant arr7 allele causes formation
of aberrant SAMs; a loss-of-function mutation in a maize ARR
homolog resulted in enlarged meristems.
3. Negative Feedback Loop between Auxin Signals and CUC
and KNOXI in the SAM
Auxin, PIN, KNOXI and CUC are expressed in mutually
exclusive domains in the SAM (Heisler et al., 2005). Loss of
maximum auxin levels in shoot tips is consistent with loss of
organogenic capacity that is caused at least partially by ectopic
expression from KNOXI. Organ initiation is partially restored in
pin1 mutants by loss of BP function (Section 1.A.) (Hay et al.,
2006). High auxin activity in lateral organ primordia, together
with AS1, represses CUC and KNOX1 expression, facilitating
lateral organogenesis (Hay et al., 2006; Heisler et al., 2005).
KNOX I proteins might also inhibit auxin transport (Treml et al.,
2005). Ectopic KNOX I expression in leaves alters auxin trans-
port and activity gradients, dramatically altering leaf shape (Hay
et al., 2006), suggesting KNOX I and auxin may function
in a feedback loop that reinforces a developmental boundary
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PLANT SHOOT ORGANOGENESIS 115
between meristem and leaf primordia. Combined regulation of
KNOX1 and auxin in boundary delimitation was also suggested
by analysis of the boundary-expressed LOB domain protein
JAGGED LATERAL ORGANS (JLO), which activates STM
and BP expression and represses expression of PIN auxin efflux
transporters (Borghi et al., 2007).
III. MOLECULAR ANALYSIS OF PLANT CELL
DIVISION
The mitotic cell division cycle consists of four sequential
phases, Gap phase 1 (G1), DNA synthetic phase (S), Gap phase
2(G
2) and mitosis (M). G1intercedes between M and entry into
the next S phase; G2separates S from M phases. Cells in G2
contain double the genetic material compared to cells in G1.The
gaps are regulatory points for cell division cycle controls that
respond to variable internal developmental and external environ-
mental factors (Dewitte et al., 2003). An alternative cell cycle
that occurs in some plant cells, the endocycle (DNA replica-
tion without mitosis), involves repeated S and G phases without
subsequent mitosis, resulting in endopolyploidy. For example,
Arabidopsis trichomes have 32C DNA content versus 2C for
diploid cells (Melaragno et al., 1993). Cell division has been
suggested to be a principal determinant of meristem activity
and overall growth rate; modulation of plant cell growth rate is
mainly achieved through regulation of G1-to-S phase transition
(Cockcroft et al., 2000).
A. Cyclin-Dependent Kinase Complexes
Large functional similarities exist between plants and ani-
mals in the core molecular mechanisms controlling cell cycle,
although plants have some unique features, e.g., cell walls and
continuous generation of new organs. As with all eukaryotes,
the cell division cycle in plants is directly controlled by Ser/Thr
kinases, i.e., cyclin-dependent kinase (CDK) complexes, which
contain a catalytic CDK subunit, a regulatory cyclin subunit
either activating or inhibiting and scaffolding proteins.
Cell status is controlled by overall levels of CDK activity. The
activity of the catalytic CDK subunit, which depends on binding
of regulatory proteins or cyclins, is responsible for recognizing
and phosphorylating a target motif present in substrate proteins;
regulatory cyclins can discriminate between distinct protein sub-
strates. Different CDK-cyclin complexes phosphorylate numer-
ous substrates at the G1-to-S and G2-to-M transitions, triggering
DNA replication and mitosis, respectively, to regulate the cell
division cycle (Dewitte et al., 2003; Menges et al., 2005; Shaul,
1996).
1. Cyclin-Dependent Kinases (CDKs)
The first CDK gene, cdc2, was identified in Schizosaccha-
romyces pombe (Hindley et al., 1984). Plant homologues were
found in pea (Feiler, 1990), Arabidopsis (Ferreira et al., 1991),
alfalfa (Hirt et al., 1992), petunia (Bergounioux et al., 1992), rice
(Hashimoto et al., 1992), maize (Colasanti et al., 1993), soybean
(Miao et al., 1993), and Antirrhinum (Fobert et al., 1994). Par-
tial complementation of yeast cell-cycle mutants proved func-
tional equivalence from those from alfalfa, maize, rice, and
soybean.
Expression of the first Arabidopsis CDK isolated, cdc2aAt
(CDKA; 1), correlated with cell division and competence for cell
division (Ferreira et al., 1991; Hemerly et al., 1993; Martinez
et al., 1992). Expression from cdc2aAt was in root and shoot
apices, young developing leaves and at the root-shoot junction
where adventitious roots initiate; it was nearly undetectable in
fully expanded leaves. In the SAM, cdc2aAt expression corre-
sponds to patterns of mitotic activity.
Unlike the single cdc2 in yeast, plants have numerous distinct
CDKs that function in different temporal and spatial phases.
The 29-member Arabidopsis CDK family is classified into
six groups, A to F, in addition to numerous CDK-like pro-
teins (Menges et al., 2005). Four of the six groups function in
cell cycle regulation. A-type CDKs, which contain a conserved
PSTAIRE motif in their cyclin-binding domain, is required for
cyclin binding (Dewitte et al., 2003; Inz´
eet al., 2006). A-type
CDKs, expressed nearly constitutively throughout the cell cy-
cle, peak at G1-to-S and G2-to-M transitions, suggesting they
function by interacting with distinct cyclins in different cell
cycle phases. B-type CDKs, unique to plants and expressed
only from S through M phases or during G2to M transition,
are activated by binding with mitotic cyclins. Two subgroups
were identified, CDKB1 present from S-phase to early M and
CDKB2 from the G2to M boundary (Menges et al., 2005). Cell
division-dependent CDKB2 expression is required for SAM ac-
tivity (Andersen et al., 2008). Function of C-type CDKs, similar
to two human proteins, and E-types, unique to plants, is largely
unknown.
CDK activity is regulated by reversible phosphorylation, per-
formed by CDK-activating kinases (CAKs); D- and F- type
CDKs act as CAKs, inducing a conformational change that al-
lows proper substrate recognition. Arabidopsis has four CAKs
in two distinct groups. (i) Cyclin H-dependent D-type CDKs
(CDKD) bind cyclin H, phosphorylate and activate both CDKs
and the C-terminal domain tail of RNA polymerase II. (ii) Plant-
specific cyclin H-independent F-type CDKs (CDKFs), specific
for CDKs only (Shimotohno et al., 2004; Yamaguchi et al.,
2003).
Activity of CAKs correlates with the cell division cycle.
Increased expression of rice nuclear CDKD1, which expresses
preferentially in S-phase, promotes S-phase progression and
overall growth rate of suspension cells (Fabian-Marwedel et
al., 2002). Overexpression of rice CDKD2 in tobacco leaf
explants caused callus formation without CK addition; callus
induction depended on CDK activation (Yamaguchi et al.,
2003). Decreasing expression from CDKF (CAK1At) caused
gradual reduction in CDK activity, arrest of cell division and
premature differentiation of root meristems in transformed
plants (Umeda et al., 2000), indicating CAKs play important
roles in determining growth rates and differentiation.
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116 L. MENG ET AL.
2. Cyclins
Plant cyclin families are complex, comprised of orthologs
from most major mammalian cyclin groups and some unique
members. Arabidopsis has at least 50 cyclins of ten types (Wang
et al., 2004) and, except for a few A-, B-, D-, and H-types, the
majority are poorly understood. In general, A- and B-types,
required for cells to enter mitosis, are termed mitotic cyclins.
A-type cyclins regulate the S-to-M phase transition; B-types
function in both G2-to-M transition and intra-M-phase control.
C-, D-, and E-types are G1-to-S phase cyclins (Lew et al., 1991).
D-type cyclins (CYCDs), the expression of which mainly
correlates with the proliferative status of cells, appear to be
the most important regulators of cell division as they aid exit
from the quiescent state (G0) and re-entry into the cell cycle
(Potuschak et al., 2001). CYCD levels respond to endogenous
and extracellular signals (Hu et al., 2000; Meijer et al., 2000;
Richard et al., 2002; Riou-Khamlichi et al., 1999). If such sig-
nals are removed, D-type cyclin levels decline rapidly, resulting
in cells remaining blocked in G1(Diehl et al., 1997). CYCDs
play key roles in G1-to-S phase transition and Arabidopsis has
10 CYCD genes (Wang et al., 2004). It appears D-type cyclins
may regulate G2-to-M transition and some D-types appear to
act as key triggers in hormonal response. Overexpression from
CYCD3;1 induces calli formation in the absence of CK (Riou-
Khamlichi et al., 1999) and expression levels of CYCD3;1 were
rate-limiting for cell division in calli induced by CK.
Transcripts for cyc1At are restricted primarily to the root api-
cal meristem (Hemerly et al., 1993). When the cyc1At promoter
was fused to GUS, a close correlation between GUS and mitotic
activity was observed in SAMs, developing flowers and embryos
(Ferreira, 1994). During de-differentiation of mesophyll proto-
plasts, cyc1At-driven GUS expression was induced only when
cell division occurred after treatment with appropriate combi-
nations of auxins and CKs. Various cyclins are expressed during
the cell cycle with each cyclin perhaps playing a different role
during cell division (Fobert et al., 1994; Hirt et al., 1992).
Based on studies of cyc1At and cdc2aAt in Arabidopsis, a
simplified model was proposed for the role of cyclins during
plant development and de-differentiation (Shaul, 1996). Cell
cycle genes are highly expressed in young, dividing tissues;
however, during differentiation reduction, but not elimination, in
cdc2aAt expression occurs, followed by cessation of cyc1At ex-
pression. In differentiated tissues, cdc2aAt expression levels re-
flect division competency. De-differentiation and re-acquisition
of division competency requires cdc2aAt activation. D-type cy-
clins mediate exit from G0and re-entry into the cell cycle. Com-
pared to A- and B-types, expression of D-types is not restricted
to dividing cells; it is expressed earlier in the cell cycle.
B. Regulation of Cell Cycle during Plant Development
To maintain normal organization and activity of a meristem
during plant development, cell division must be tightly con-
trolled by machinery that regulates the cell cycle and is linked
directly to SAM activity.
1. Genetic Factors Regulate Plant Cell Cycle and Meristem
Activity
Cell cycle regulation involves transcriptional activation of D-
type cyclin genes. AINTEGUMENTA (ANT) encodes an AP2-
domain transcription factor found only in plants that is expressed
on flanks of the SAM at sites of developing leaf primordia. Loss
of function ant mutants form smaller leaves. Plants overexpress-
ing ANT form larger leaves and this correlates with induction of
cell cycle genes and promotion of cell proliferation (Mizukami
et al., 2000).
In plants, Myb proteins control G2-to-M transition by acti-
vating or repressing transcription. MYB3R1 and MYB3R4 play
partially redundant roles in positively regulating cell division.
Double mutants of myb3r1/myb3r4 often fail to complete cell
division, which correlates with transcript reduction from sev-
eral G2-to-M phase-specific genes (Haga et al., 2007). A Myb-
related cell-division-cycle transcription factor, AtCDC5, may
be essential for G2-to-M transition, and may regulate SAM ac-
tivity by controlling expression from STM and WUS (Lin et al.,
2007).
Arabidopsis PASTICCINO (PA S ) encodes protein phos-
phatases, possibly involved in cell division regulation in the
SAM in response to phytohormones (Faure et al., 1998; Harrar
et al., 2003). Cells in pas mutants are more competent for cell
division, demonstrated in the CK hypersensitive response and
the ectopic cell division in the SAM. Expression of CDKA and
CYCB1 and certain KNOX I genes upregulate STM, KNAT2 and
KNAT6 in pas mutants.
2. Exogenous Signals Regulate the Plant Cell Division Cycle
Upstream signaling components of the cell cycle are less
well characterized but appear to involve exogenous signals like
phytohormones (particularly, CK and auxin), sugars and stress.
CycD3, elevated in a mutant with high CK levels, was rapidly in-
duced by CK application in both cell cultures and whole plants.
Constitutive expression of CycD3 in transgenic plants leads to
induction and maintenance of cell division without exogenous
CK addition. CK activates cell division through induction of
CycD3 at the G1-to-S transition (Riou-Khamlichi et al., 1999).
Auxin is also thought to promote cell cycle activity by trigger-
ing degradation of inhibitory proteins that induce expression of
genes involved in G1-to- S and G2-to-M transitions (Blilou et
al., 2002; Hartig et al., 2006).
Using a cdc2aAt promoter-GUS fusion, GUS expression in
mesophyll protoplasts from leaves was studied during dedif-
ferentiation (Hemerly et al., 1993). Cultivation of protoplasts
in the presence of auxin or CK caused cdc2aAt-mediated in-
duction of GUS expression. Despite lack of cell division, divi-
sion competence was likely due to hormonal effects on endoge-
nous cdc2aAt. Consistent with this, there was rapid induction
of cdc2aAt promoter-driven GUS expression around damaged
surfaces of wounded transgenic Arabidopsis leaves.
Possible links have been identified between CK and cell di-
vision, possibly in the G2-to-M phase. In tobacco protoplasts
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PLANT SHOOT ORGANOGENESIS 117
CK controls the cell cycle at mitosis by stimulating Tyr dephos-
phorylation and activation of the p34cdc2-like H1 histone kinase
(Zhang et al., 1996). CK also regulates G1-to-S transition, me-
diated by CycD3 (Riou-Khamlichi et al., 1999). Application
of CK to both seedlings and in vitro cultured cells resulted in
increased steady-state levels of CycD3 mRNA. With regard to
in vitro plant development, leaf explants overexpressing CycD3
form healthy green calli without CK in contrast to wild-type
explants that form such calli only with addition of CK. This
suggests that CycD3 overexpression bypasses CK in activating
the cell cycle; CK appears to regulate the cell cycle through
CycD3.
CK transport through tissues may also play a role in the in
vitro CK response since cells responding during in vitro shoot
organogenesis often are not in direct contact with hormones in
the media. During in vitro culture of vegetative shoots of maize
and barley on a medium with a high CK to auxin ratio, respond-
ing cells were in axillary shoot meristematic domes (Zhang
et al., 1998) or nodal regions (Zhang et al., 2002), neither of
which were in direct contact with hormone-containing medium,
while cells at the cut edge of the stem in direct contact with the
medium did not respond. The possible role of a CK transporter
is further supported by studies of its role during plant growth.
CK exists in the xylem sap while the root tip is its major site
of biosynthesis, suggesting that CK is transported through the
xylem to aerial parts of a plant. Identification of a putative CK
transporter, AtPUP1, was based on functional complementation
of a yeast mutant deficient in adenine uptake (Gillissen et al.,
2000).
IV. MOLECULAR ANALYSIS OF
IN VITRO
SHOOT
ORGANOGENESIS
A. Use of Specific Genes as Molecular Markers
Historically, study of in vitro shoot organogenesis was based
on morphological or physiological observations. With cloned
genes available, molecular markers can now be used to develop
a better molecular understanding of in vitro shoot organogen-
esis. The first example of this approach used expression anal-
ysis of maize KN1 and its homolog in barley during in vitro
axillary shoot meristematic cell proliferation and adventitious
shoot meristem formation (Zhang et al., 1998). Vegetative shoot
segments from germinated maize and barley seedlings were cul-
tured in vitro on a high CK, low auxin medium. Within weeks,
cell division in the cultured axillary shoot meristems changed
from a well-regulated state to a proliferating state, with the small
meristematic dome becoming enlarged. Adventitious meristems
(ADMs) arose directly from cells in the enlarged dome (Fig.
3A). Expression of KN1 was maintained in shoot meristematic
cells during in vitro cell proliferation of axillary shoot meristems
(Fig. 3B). ADMs appear to derive directly from KN1-expressing
shoot meristematic cells. Thus, KN1 can detect in vitro shoot
meristem formation that appears to follow paths similar to in
planta shoot meristem development.
More recently, CUC2 and WUS were shown to be the earli-
est molecular markers for in vitro shoot organogenesis. Both are
expressed in a small number of progenitor cells, deemed com-
petent for new shoot meristem initiation (Gordon et al., 2007).
Dynamic fine-tuning of CUC2 and WUS expression and a lo-
cal CK and auxin gradient feedback loop have been suggested
to lead to a self-organizing cell identity partition that gradu-
ally establishes shoot meristem cell niches within callus tissue.
Subsequent patterning and development of the shoot meristems,
once shoot premeristems are initiated, involves local activation
of genes expressed in early shoot meristems, e.g., PIN1, STM,
REV, FIL, ATML1 and CLV3, thought to be largely autonomous.
CUC1 and CUC2 genes are required for embryonic SAM for-
mation and cotyledon separation (Aida et al., 1997). Mutations
in CUC1 and CUC2 or CUC1 alone reduce initiation efficiency
of adventitious shoots during in vitro culture (Aida et al., 1997;
Daimon et al., 2003; Takada et al., 2001), while overexpres-
sion increases adventitious shoot formation on calli; CUC1 and
CUC2 activate STM expression in calli (Daimon et al., 2003).
CUC2 expression marks a small number of progenitor cells that
FIG. 3. Expression of KNOTTED1 in the in vitro–generated adventitious shoot meristems (ADMs) of maize. (A) Multiple ADMs induced from an enlarged
shoot meristematic dome in vitro. (B) Expression of KNOTTED1 (arrow) in the in vitro ADMs. ). (From Trigiano, R. N. and Gary, D. J., Eds., Plant Tissue Culture
and Development, CRC Press LLC, Boca Raton, FL, 2004)
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118 L. MENG ET AL.
proliferate to form a relatively homogeneous cell mass that later
patterns into a de novo shoot meristem.
WUS expression in calli is essential to initiate shoot meristem
progenitor cell identity; continuous WUS expression is required
for further shoot meristem development. A strong wus mutant
(wus-1) generated only 5% as many shoots as wild-type (Gordon
et al., 2007). Ectopic WUS expression induces shoot generation
in roots (Gallois et al., 2004). Moreover, a mutant in which
WUS expression is elevated causes somatic embryo formation
in a range of tissues and organs (Zuo,etal., 2002), suggest-
ing that WUS is sufficient to establish shoot meristem identity.
Therefore, CUC2 and WUS can be used as molecular markers
for shoot premeristem progenitor cells, whereas PIN1,STM,
REV,FIL,ATML1and CLV3, can be used to identify developing
shoot meristems during in vitro shoot organogenesis.
Using genes as molecular markers provides new insights
into in planta and in vitro plant development. They will
continue to increase our understanding of these complex
processes as additional genes and networks are identified and
characterized. These studies continue to validate the hypothesis
that de novo organogenesis involves three steps: acquisition of
competence, shoot induction, and organogenesis determination
(Christianson, 1985).
B. Identification of Genes Involved in
In Vitro
Shoot
Organogenesis
As described earlier in this review many regulatory factors
and pathways are involved in controlling formation and devel-
opment of the SAM in planta and in vitro. Besides the key
pathways of CUC1/CUC2-STM and WUS-CLV3, other impor-
tant regulators have also been identified by genetic analyses of
SAM-defective mutants. These studies reveal a complex and
elegant regulation network involved in embryonic formation of
the SAM and in adventitious shoot organogenesis.
Genetic and molecular efforts are now used to identify genes,
which might either regulate or be involved directly in shoot
organogenesis in vitro. For example, a novel MADS box cDNA,
PkMADS1, was isolated from a cDNA library of leaf explants
from a woody tree species, Paulownia kawakamii, which un-
dergoes adventitious shoot formation (Prakash et al., 2002).
Deduced amino acid sequence of its MADS domain revealed
90% homology to the Arabidopsis AGL24 (AGAMOUS-like)
protein (Hartmann et al., 2000) and to the STMADS16 protein in
potato (Carmona et al., 1998). Expression from PkMADS1 was
not detected in callus cultures of Paulownia, but was in shoot-
forming cultures. In planta,PkMADS1 transcripts were found
only in shoot apices, not in root apices, flowers or leaf explants.
Plants with antisense knockouts of the gene had stunted shoots,
altered phyllotaxy and in some cases lacked SAM development.
Shoot regeneration from leaf explants of antisense plants was
reduced ten-fold versus wild-type or PkMADS1 overexpressing
plants, suggesting PkMADS1 expression is essential for in vitro
and in planta shoot formation.
Because CKs are the most efficient growth regulators that
induce in vitro shoot organogenesis, genes involved in CK
metabolism or signal transduction are likely to affect shoot
organogenesis. When ESR1 (ENHANCER OF SHOOT RE-
GENERATION1), which allows for CK-independent shoot in-
duction (Banno et al., 2001), is overexpressed, this putative tran-
scription factor greatly enhanced shoot regeneration efficiency
following CK addition to root explants, coupled with a reduction
in the optimal CK concentration needed (Buttner et al., 1997;
Fujimoto et al., 2000). Also wild-type root explants, cultured
on CK-containing shoot-induction medium, had transiently el-
evated ESR1 transcript levels, which occurred before STM ex-
pression but after acquisition of competence for shoot regener-
ation. These results suggest that ESR1 may be a downstream
effecter for CK that enhances shoot regeneration initiation after
acquisition of competence for shoot organogenesis. Inter-
estingly, inducible overexpression from ESR1 leads to CK-
independent shoot regeneration on roots. Constitutive ESR1
overexpression, however, results in dark green calli with no
shoot development (Banno et al., 2001). These results suggest
an ESR1 gradient and/or other factors may be necessary for
shoot development during in vitro organogenesis.
Besides ESR1, a homologue, ESR2, is involved, and even
more active than ESR1, in promoting shoot regeneration in tis-
sue culture. Overexpression of ESR2 results in CK-independent
shoot regeneration from cre1/ahk4/wol mutant roots and res-
cues cre1/ahk4/wol mutants, suggesting ESR2 is an important
regulator in the CK response. ESR2 plays a role in shoot re-
generation through transcriptional regulation of CUC1 based
on an ESR2 knockdown that downregulates CUC1 expression
and phenocopies the cuc1 mutant (Aida et al., 1997). ESR2 also
activates CYCD1;1,AHP6, and CUC1 expression (Ikeda et al.,
2006).
Two genes, RGD3 (ROOT GRORWTH DEFECTIVE3) and
RID3 (ROOT INITIATION DEFECTIVE3), were shown to con-
trol cell division in the SAM during shoot organogenesis from
hypocotyl explants during in vitro culture. RID3, a negative
regulator, and RGD3, a positive regulator, of the CUC-STM
pathway participate in the proper control of cell division in the
SAM. RGD3 is expressed in the developing SAM, while RID3
is expressed outside the SAM in the early stages of shoot regen-
eration (Tamaki et al., 2009).
V. SUMMARY
Molecular and genetic analyses of in planta and in vitro shoot
organogenesis to date lend support to the developmental model
proposed by Christianson and Warnick (1985), namely that
shoot organogenesis is divided into three phases: competence
acquisition, i.e., the stage before cells are induced to develop
into a shoot, shoot induction, and shoot development. Expres-
sion from genes involved in the CK signal transduction pathway,
e.g., CKI1 and CRE1, might be indicators of cell competence
to respond to CK, normally required for shoot organogenesis.
Downloaded by [University of California, Berkeley] at 17:11 16 August 2013
PLANT SHOOT ORGANOGENESIS 119
The best candidates as indicators of cell division competence
are likely CDC2a-and cyclin-type genes, induced during shoot
organogenesis from stem tissues (Boucheron et al., 2002). For
the shoot induction phase, maize KN1-type homologues appear
to be the most definitive molecular markers to identify early
stages of induction of in vitro shoot organogenesis.
CK likely acts through activation of CycD-3 type genes to
initiate or accelerate the cell cycle; however, the molecular con-
nection between CK action and CycD-3 activation has not yet
been fully elucidated. Although molecular insights already exist
as to why a higher ratio of CK to auxin favors shoot organogen-
esis, the precise molecular pathway still is not known. Future
study of genes and regulatory pathways involved in dediffer-
entiation of somatic cells in vitro, coupled with the elucidation
of the role of chromatin structure changes during the course
of dedifferentiation, will provide the insights needed to under-
stand fully in vitro shoot organogenesis and the developmental
flexibility of the cell in planta.
ACKNOWLEDGMENTS
We dedicate this review to the memory of Dr. Shibo Zhang,
whose life goal was to understand the molecular pathways in-
volved in in vitro shoot development. Although his death in a
tragic accident prevented completion of his dream, we are sure
that he would be pleased with the progress made in this field.
We are grateful to Barbara Alonso for helping to prepare the
manuscript. We attempted to cite all pertinent work but apolo-
gize for any citations that were inadvertently missed.
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