Review
Leaf adaxial–abaxial polarity specification and lamina
outgrowth: evolution and development
Takahiro Yamaguchi*, Akira Nukazuka and Hirokazu Tsukaya
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan
*Corresponding author: E-mail, [email protected]; Fax/Tel, +81-3-5841-4047.
(Received February 20, 2012; Accepted May 10, 2012)
A key innovation in leaf evolution is the acquisition of a flat
lamina with adaxial–abaxial polarity, which optimizes the
primary function of photosynthesis. The developmental
mechanism behind leaf adaxial–abaxial polarity specification and flat lamina formation has long been of interest to
biologists. Surgical and genetic studies proposed a conceptual model wherein a signal derived from the shoot apical
meristem is necessary for adaxial–abaxial polarity specification, and subsequent lamina outgrowth is promoted at the
juxtaposition of adaxial and abaxial identities. Several distinct regulators involved in leaf adaxial–abaxial polarity specification and lamina outgrowth have been identified.
Analyses of these genes demonstrated that the mutual
antagonistic interactions between adaxial and abaxial determinants establish polarity and define the boundary between
two domains, along which lamina outgrowth regulators
function. Evolutionary developmental studies on diverse
leaf forms of angiosperms proposed that alteration to the
adaxial–abaxial patterning system can be a major driving
force in the generation of diverse leaf forms, as represented
by ‘unifacial leaves’, in which leaf blades have only the
abaxial identity. Interestingly, unifacial leaf blades become
flattened, in spite of the lack of adaxial–abaxial juxtaposition. Modification of the adaxial–abaxial patterning system
is also utilized to generate complex organ morphologies,
such as stamens. In this review, we summarize recent advances in the genetic mechanisms underlying leaf adaxial–
abaxial polarity specification and lamina outgrowth, with
emphasis on the genetic basis of the evolution and diversification of leaves.
Keywords: Adaxial–abaxial polarity Development
Evolution Lamina outgrowth Leaf Unifacial leaf.
Abbreviations: ARF, auxin response factor; HD-ZIPIII, class III
homeodomain–leucine zipper; KAN, kanadi; KNOX, knottedlike homeobox; SAM, shoot apical meristem; ta-siRNA, transacting short-interfering RNA; WOX, wuschel-related
homeobox.
Introduction
The development of a flat lamina is a key event in leaf evolution,
enabling maximum light capture. A flat lamina also facilitates
differentiation of two specialized leaf domains: adaxial and
abaxial. Although there are exceptions, such as unifacial or
equifacial leaves (see below), the adaxial (upper) domain of
the leaf consists of an epidermis with a relatively thick cuticle
and densely packed layer of palisade mesophyll cells, which
optimize light capture. The abaxial (lower) domain of the leaf
consists of an epidermis with abundant stomata and spongy
mesophyll cells, which function in gas exchange and the regulation of transpiration. The leaf vasculature is also aligned along
the adaxial–abaxial axis, with xylem tissue differentiating adaxially and the phloem abaxially.
Fossil records indicate that primitive land plants had naked
branched stems with sporangia but no leaves. Leaves subsequently evolved at least twice in the evolution of land plants,
as represented by microphyllous leaves in lycophytes (e.g. the
genera Selaginella and Isoetes) and megaphyllous leaves in
euphyllophytes (ferns, gymnosperms and angiosperms)
(Boyce and Knoll 2002, Glifford and Foster 1989, Kenrick and
Crane 1997). Paleontological evidence also indicates the independent evolution of megaphyllous leaves within fern and seed
plant lineages, since seed plants evolved from leafless progymnosperms (Beck 1966, Kenrick and Crane 1997, Scheckler and
Banks 1971). Microphyllous leaves have a single vascular strand
with no leaf gap and are thought to have evolved from the
vascularization of spine-like outgrowth on the stem (the enation theory) (Bower 1935) or from the sterilization of sporangia (the sterilization theory) (Kenrick and Crane 1997). In
contrast, megaphyllous leaves are characterized by laminae
with complex venation patterns and their leaf traces are associated with the formation of leaf gaps in the stem vascular
cylinder. Megaphyllous leaves are thought to have evolved
from lateral shoot systems through a series of transitions
(Beerling and Fleming 2007, Sanders et al. 2007, Sanders
et al. 2009, Zimmerman 1952). That is, the leaf evolved from
Plant Cell Physiol. 53(7): 1180–1194 (2012) doi:10.1093/pcp/pcs074, available online at www.pcp.oxfordjournals.org
! The Author 2012. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Plant Cell Physiol. 53(7): 1180–1194 (2012) doi:10.1093/pcp/pcs074 ! The Author 2012.
Adaxial–abaxial polarity and lamina outgrowth
an indeterminate radial structure to one that is determinate,
dorsoventral and laminar. Each transition is thought to have
involved a novel genetic mechanism.
Recent molecular genetic studies of model angiosperms
have greatly expanded our knowledge of the genetic mechanisms underlying leaf adaxial–abaxial polarity specification and
flattened lamina formation. These studies have also provided
clues about the genetic mechanisms behind the origin and
diversification of leaves. In this review, we summarize recent
advances in the genetic regulation of leaf adaxial–abaxial
polarity specification and leaf flattening. We also discuss the
genetic basis of the evolution and diversification of leaves.
Separation
from the SAM
A
Ab
Ad
Abaxialized
radial leaf
Signal
Lamina
outgrowth
Lamina
outgrowth
Ad
Ab
B
Adaxial
A model of adaxial–abaxial polarity
specification and lamina outgrowth
In plants, cell fate is determined mainly by positional information rather than cell lineage. Since leaf primordia develop from a
group of cells on the flank of the shoot apical meristem (SAM),
leaves possess inherent positional relationships with the SAM:
the adaxial side of leaf primordia is derived from cells adjacent
to the SAM, while the abaxial side is derived from more distant
cells (Fig. 1A). Microsurgical studies by Sussex in the 1950s
(Sussex 1951, 1955) demonstrated that communication
between the SAM and leaf primordia is required for the establishment of leaf adaxial–abaxial polarity. When young or incipient leaf primordia are isolated from the SAM, leaves are
radialized with only abaxial characteristics. Later refinement
of these surgical experiments using laser ablation and microdissection also resulted in radialized leaf development with only
abaxial identity (Reinhardt et al. 2005). These experiments
demonstrate that a signal from the SAM is necessary for specification or maintenance of adaxial identity, with leaves assuming abaxial identity in the absence of adaxial identity.
Furthermore, the result also suggests that abaxial identity
alone is not sufficient for lamina outgrowth.
How adaxial–abaxial leaf polarity and lamina outgrowth are
related was defined from phenotypic analysis of the phantastica
(phan) mutant in Antirrhinum majus (Waites and Hudson
1995). In phan mutants, leaves show varying degrees of abaxialization. Leaves that exhibit a weak phan phenotype have
ectopic patches of abaxial tissues on the adaxial side, with adventitious lamina around these patches. Leaves that exhibit a
strong phan phenotype are fully abaxialized and fail to expand
laterally, resulting in radialized leaves (Fig. 1B). These phenotypes indicate that PHAN promotes leaf adaxial identity in
A. majus. Furthermore, Waites and Hudson (1995) proposed
a hypothesis whereby lamina outgrowth is promoted at
the juxtaposition between adaxial and abaxial identities.
Complete abaxialization leads to loss of the adaxial–abaxial
boundary, resulting in radialized leaves, while ectopic abaxial
patches on the adaxial side lead to an ectopic adaxial–abaxial
boundary, which drives ectopic lamina outgrowth. The ‘adaxial–abaxial juxtaposition hypothesis’ has been verified in a
Abaxial
Abaxialized
Wild-type leaf
phan mutant leaf
Fig. 1 Adaxial–abaxial polarity specification and lamina outgrowth.
(A) Schematic of the shoot apex as viewed from above. An incipient
leaf primordium (dashed circle) develops from the flanks of the SAM
and perceives the adaxialization signal from the SAM. Separation of an
incipient leaf primordium from the SAM results in abaxialized radial
leaf development. During leaf development, the adaxial domain (ad)
differentiates adjacent to the SAM, whereas the abaxial domain (ab)
differentiates away from the SAM. Lamina outgrowth is promoted at
the juxtaposition between the adaxial and abaxial domains. (B) A
flattened leaf in the wild-type (left) and an abaxialized radial leaf in
the phan mutant (right) of A. majus. Images in (B) are reproduced
from Waites and Hudson 1995; Development 121: 2143–2154.
Copyright ! The Company of Biologists.
number of subsequent studies on mutants or transgenic
plants with abnormal leaf adaxial–abaxial polarity, as reviewed
below. Interestingly, the system of outgrowth at the boundary
seems to be often utilized in the development of multicellular
organisms, because an analogous system is observed in the appendage outgrowth along the dorsoventral boundary in animals, such as in the development of insect wings and vertebrate
limbs (Grimm and Pflugfelder 1996).
Adaxial determinant: ARP family
The PHAN gene and orthologs in other species encode MYB
transcription factors referred to as the ARP family [from
ASYMMETRIC LEAVES1 (AS1), ROUGH SHEATH2 (RS2) and
PHAN] (Byrne et al. 2000, Pozzi et al. 2001, Timmermans
et al. 1999, Tsiantis et al. 1999, Waites et al. 1998). In these
species, ARP genes are uniformly expressed in young leaf primordia, suggesting that their role in adaxial fate specification is
Plant Cell Physiol. 53(7): 1180–1194 (2012) doi:10.1093/pcp/pcs074 ! The Author 2012.
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regulated by interacting protein partners. In Arabidopsis thaliana (Arabidopsis), AS1 forms a protein complex with
ASYMMETRIC LEAVES2 (AS2) (Guo et al. 2008, Yang et al.
2008). AS2 encodes a plant-specific AS2/LOB domain protein
containing a leucine-zipper motif and is expressed in the adaxial
surface of leaf primordia (Iwakawa et al. 2007, Iwakawa et al.
2002, Shuai et al. 2002). Although neither as1 or as2 single
mutants nor the as1 as2 double mutant show obvious adaxial–abaxial polarity defects, overexpression of AS2 results in
adaxialized leaf development, suggesting that AS1/AS2 acts redundantly with other pathways in adaxial fate specification
(Iwakawa et al. 2007, Lin et al. 2003). Indeed, adaxial–abaxial
polarity defects in as1 and as2 are enhanced by a number of
mutations, e.g. in genes of ribosomal proteins, components of
the 26S proteasome, biogenesis of trans-acting short-interfering
RNA (ta-siRNA) and chromatin modification (Garcia et al.
2006, Horiguchi et al. 2011, Huang et al. 2006, Kojima et al.
2011, Li et al. 2005, Ori et al. 2000, Phelps-Durr et al. 2005,
Pinon et al. 2008, Szakonyi and Byrne 2011, Ueno et al. 2007,
Xu et al. 2003, Xu et al. 2006, Yang et al. 2006, Yao et al.
2008). In maize (Zea mays), it is not obvious whether RS2 is
involved in adaxial fate specification, but the INDETERMINATE
GAMETOPHYTE1 gene, which encodes an ASL/LOB domain
protein similar to AS2 in Arabidopsis, plays a role in adaxial–
abaxial polarity specification (Evans 2007), suggesting partial
conservation of the AS1/AS2 pathway in leaf adaxial fate
specification.
One conserved role of ARP is to repress the expression of
class I KNOTTED-LIKE HOMEOBOX (KNOX) genes within
developing leaf primordia. The mutual antagonistic relationship
between ARP and KNOX is important in promoting determinacy in angiosperm leaves (Byrne et al. 2000, Ori et al. 2000,
Semiarti et al. 2001). In plants with simple leaves, expression
of KNOX genes is down-regulated in leaf primordia throughout
their development (Jackson et al. 1994, Lincoln et al. 1994). In
contrast, in most plants with compound leaves, KNOX genes
are transiently down-regulated in incipient leaf primordia, but
become expressed in developing leaf primordia (Bharathan
et al. 2002, Hareven et al. 1996). By a series of loss- and
gain-of-function studies, it has been revealed that reactivation
of KNOX genes in developing leaf primordia is associated with
the development of compound leaves (Hareven et al. 1996,
Hay and Tsiantis 2006, Koenig and Sinha 2010, Uchida et al.
2010). In the lycophyte Selaginella kraussiana, an ARP ortholog
is expressed in microphyll primordia, whereas KNOX homologs
are down-regulated (Harrison et al. 2005). Furthermore, an
ARP ortholog from S. kraussiana can rescue defects of the as1
mutant in Arabidopsis (Harrison et al. 2005). Thus, the antagonistic mechanism between ARP and KNOX may have been
independently co-opted to promote leaf determinacy within
lycophytes and seed plant lineages from a leafless common
ancestor. In several ferns, KNOX genes are not down-regulated
in incipient leaf primordia (Bharathan et al. 2002, Sano et al.
2005). This result is in agreement with the independent origin
of leaves within ferns and seed plants, and may reflect the
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different developmental characteristics of fern leaves, in
which extensive apical growth occurs by means of an apical
cell (Bower 1884).
Adaxial determinant: HD-ZIPIII family
Several distinct families of transcription factors play key roles in
the establishment of leaf adaxial–abaxial polarity with polar
expression patterns. The class III HOMEODOMAIN–LEUCINE
ZIPPER (HD-ZIPIII) gene family plays a key role in adaxial fate
determination. HD-ZIPIII proteins contain an N-terminal
DNA-binding homeodomain and leucine-zipper motif that
facilitates protein dimerization, a putative lipid or steroid binding START domain and a C-terminal PAS-like MEKHLA domain,
which may be involved in chemical sensing (Magnani and
Barton 2011, McConnell and Barton 1998, McConnell et al.
2001, Otsuga et al. 2001). HD-ZIPIII genes also contain a binding site for microRNAs, miR165 and miR166 (Emery et al. 2003,
Rhoades et al. 2002, Tang et al. 2003). The Arabidopsis
genome contains five HD-ZIPIII genes: PHABULOSA (PHB),
PHAVOLUTA (PHV), REVOLUTA (REV), ATHB8 and ATHB15
(also known as CORONA and INCUVATA4). PHB, PHV and
REV belong to a single clade (the REV clade) and are expressed
in the adaxial domain of developing leaf primordia, in the central zone of the SAM and in the xylem pole of the vasculature,
while ATHB8 and ATHB15 are exclusively expressed in vascular
tissues. Simultaneous loss-of-function of PHB, PHV and REV
results in abaxialized cotyledons (Emery et al. 2003, Prigge
et al. 2005). Conversely, gain-of-function mutations of PHB
and PHV, which disrupt the microRNA-binding site, result in
ectopic expression throughout leaf primordia and the development of adaxialized radial leaves (McConnell and Barton 1998,
McConnell et al. 2001). Both loss- and gain-of-function mutant
phenotypes indicate that HD-ZIPIII genes are necessary and
sufficient for the specification of leaf adaxial identity. In maize
and rice (Oryza sativa), the REV clade genes are expressed in the
adaxial domain of leaf primordia and ectopic expression results
in the development of adaxialized leaves (Itoh et al. 2008,
Juarez et al. 2004a, Juarez et al. 2004b). Thus, the function of
the REV clade of HD-ZIPIII genes in adaxial fate specification
appears to be conserved among angiosperms.
Abaxial determinant: KANADI family
Several families of transcription factors promote abaxial fate
specification. Members of the KANADI (KAN) gene family,
which encode GARP-domain transcription factors, are
expressed in the abaxial domain of leaf primordia and in the
phloem of the vasculature, in a pattern complementary to that
of the REV clade of HD-ZIPIII genes (Eshed et al. 2001, Izhaki
and Bowman 2007, Kerstetter et al. 2001). In Arabidopsis, individual loss-of-function mutants of KAN (KAN1–KAN4) show
weak or no phenotypes in leaves; however, progressive loss of
KAN activity in double (kan1 kan2) and triple (kan1 kan2 kan3)
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Adaxial–abaxial polarity and lamina outgrowth
mutants results in dramatic adaxialization of leaves with loss of
lamina flattening, ranging from fully radialized leaves to narrow
leaves with ectopic adaxial sectors on the abaxial side (Eshed
et al. 2001, Eshed et al. 2004). Conversely, ectopic expression
of KAN1 or KAN2 throughout the leaf primordia results in
abaxialized radial leaves (Eshed et al. 2001). The expression
patterns and loss- and gain-of-function mutant phenotypes
indicate that KAN is necessary and sufficient for abaxial identity.
In the Japanese morning glory, Ipomoea nil, loss of KAN activity
by the feathered mutation results in partial leaf abaxialization
(Iwasaki and Nitasaka 2006). In monocots, mutations in the
maize KAN gene, milkweed pod1, also cause ectopic adaxial
tissue formation on the abaxial side with concomitant
mis-expression of the HD-ZIPIII gene in the abaxial side
(Candela et al. 2008). Similarly, mutations in the rice KAN
gene, SHALLOT-LIKE1, result in partial leaf adaxialization
(Zhang et al. 2009). Thus, the role of KAN in abaxial fate specification is probably conserved among angiosperms.
Abaxial determinant: AUXIN RESPONSE
FACTORS
The auxin response factors AUXIN RESPONSE FACTOR 3/ETTIN
(ARF3/ETT) and ARF4 are also involved in leaf abaxial fate specification (Pekker et al. 2005). Loss of either ARF3 or ARF4 activity does not cause severe defects in leaf adaxial–abaxial
polarity, but combined loss of these two genes results in abaxialized leaves that strikingly resemble those in the kan1 kan2
double mutant. The expression domain of ETT and ARF4 overlaps in the abaxial domain of leaf primordia, consistent with
their role in abaxial fate specification. Loss of ARF3 function
suppresses the abaxialization effect of ectopic KAN1 expression,
suggesting that ARF3/ARF4 act downstream of KAN. However,
neither ARF3 nor ARF4 expression is changed in the kan1 kan2
double mutant (Pekker et al. 2005). On the other hand, the
KAN protein physically interacts with the ARF3 protein (Kelley
et al. 2012), suggesting the possibility that KAN proteins
modulate activity or stability of ARF3/ARF4 proteins through
direct interaction. ETT and ARF4 are negatively regulated by a
small RNA known as tasiR-ARF (ta-siRNA that targets ARF3/
ARF4 transcripts), which are derived from non-coding TAS3
precursor transcripts (Allen et al. 2005, Fahlgren et al. 2006,
Hunter et al. 2006). Together with the fact that HD-ZIPIII genes
are negatively regulated by microRNAs, miR165 and miR166,
regulation of expression domains of both adaxial and abaxial
determinants by small RNAs is therefore an important aspect of
leaf adaxial–abaxial polarity specification (for review, see
Husbands et al. 2009, Kidner and Timmermans 2010).
Antagonistic interactions between adaxial and
abaxial determinants
The regulatory network controlling adaxial–abaxial polarity is
based on the mutual antagonistic interactions between adaxial
and abaxial determinants (Fig. 2). The adaxial determinant
HD-ZIPIII has mutually antagonistic interactions with the abaxial determinant KAN. In the kan1 kan2 kan3 triple mutant, the
HD-ZIPIII genes are ectopically expressed throughout radialized
leaves (Eshed et al. 2004). Conversely, ectopic expression of
KAN1 or KAN2 throughout the leaf primordia results in abaxialized radial organs, with a concomitant loss of HD-ZIPIII expression (Eshed et al. 2004, Kerstetter et al. 2001). Thus, the
KAN genes negatively regulate HD-ZIPIII expression. This negative regulation is bi-directional, because the phenotype of abaxialized cotyledons in the phb phv rev triple mutant is partially
suppressed by loss of KAN1, KAN2 and KAN4. This result suggests that HD-ZIPIII genes also negatively regulate KAN expression (Izhaki and Bowman 2007). The opposing effects of
HD-ZIPIII and KAN seem to be indirect, and in the case of vascular patterning, these components act by affecting the canalization of auxin flow (Ilegems et al. 2010).
Further mutual antagonism exists between AS2 and KAN.
KAN protein down-regulates AS2 expression in the abaxial
domain of the leaf through direct interaction with a cis-element
in the promoter region of AS2 (Wu et al. 2008). Conversely,
overexpression of AS2 results in adaxialized leaf development
Lamina
outgrowth
AS1
TAS3
HD-ZIPIII
ARP
Adaxial
domain
tasiR-ARF
PRS
WOX1
YABBY
miR165/166
KAN
ARF3
ARF4
Abaxial
domain
Auxin
Fig. 2 A genetic network for leaf adaxial–abaxial polarity specification and lamina outgrowth. Adaxial–abaxial polarity is established through
antagonistic interactions between adaxial and abaxial determinants, which include distinct families of transcriptional regulators and small RNAs.
Lamina outgrowth is promoted by several factors in response to the adaxial–abaxial juxtaposition.
Plant Cell Physiol. 53(7): 1180–1194 (2012) doi:10.1093/pcp/pcs074 ! The Author 2012.
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T. Yamaguchi et al.
reminiscent of the kan1 kan2 double mutant with reduced
levels of KAN expression, whereas expression of KAN is elevated
in the as2 mutant (Iwakawa et al. 2007, Lin et al. 2003). Thus,
AS2 and KAN negatively regulate each other. These mutual
antagonistic relationships between adaxial and abaxial determinants likely play a fundamental role in the establishment and
maintenance of leaf polarity by preventing cells from simultaneously exhibiting adaxial and abaxial identity throughout leaf
development. The mutual antagonism can also create and stabilize the boundary between two domains, along which lamina
outgrowth regulators function.
Co-option of vascular patterning in leaf
adaxial–abaxial patterning
In addition to leaves, the antagonistic relationship between
HD-ZIPIII and KAN is also involved in the radial patterning of
the stem vasculature. HD-ZIPIII and KAN genes show complementary expression patterns in the vasculature as well as in
leaves. In the stem, the xylem lies internally relative to the
phloem. HD-ZIPIII genes are expressed in the developing
xylem, whereas KAN genes are expressed in the developing
phloem (Baima et al. 1995, Emery et al. 2003, Kang and
Dengler 2002, Ohashi-Ito et al. 2002). Multiple loss-of-function
of KAN and gain-of-function of REV, PHB and PHV results in
amphivasal (xylem surrounds the phloem) vascular bundles.
Conversely, multiple loss-of-function of HD-ZIPIII genes results
in amphicribal (phloem surrounds the xylem) vascular bundles
(Emery et al. 2003, Ilegems et al. 2010, McConnell and Barton
1998, McConnell et al. 2001, Zhong and Ye 2004). Thus, antagonistic activity of HD-ZIPIII and KAN regulates the radial patterning of the stem vasculature. Since the evolution of the
vasculature predates that of seed plant leaves, it is hypothesized
that the genetic interaction between HD-ZIPIII and KAN played
ancestral roles in the radial patterning of the vasculature in
early land plants, and were then co-opted into leaf adaxial–
abaxial patterning (Emery et al. 2003, Floyd and Bowman
2010). In line with this hypothesis, expression of HD-ZIPIII
genes in the vasculature is conserved in the lycophyte
S. kraussiana (Floyd and Bowman 2006). On the other hand,
expression of HD-ZIPIII genes in the adaxial leaf domain is
observed in gymnosperms and angiosperms, but not in
S. kraussiana, suggesting that the role of HD-ZIPIII genes in
leaf adaxial fate specification was recruited during the evolution
of seed plants (Floyd and Bowman 2006). Consistent with this
idea, the role of HD-ZIPIII genes in leaf polarity is restricted to
REV clade genes, which is likely to have arisen by gene duplications that occurred just before the diversification of seed plants
(Floyd and Bowman 2006, Floyd and Bowman 2007, Prigge and
Clark 2006).
Homologs of HD-ZIPIII genes are found in the charophycean
algae Chara corallina (Floyd et al. 2006) and homologs of KAN
genes are found in the moss Physcomitrella patens (Floyd and
Bowman 2007). These findings indicate that the more ancestral
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role for HD-ZIPIII and KAN genes is not the patterning of vascular tissues, as the vascular tissues evolved after the divergence
of the moss lineage. It is possible that HD-ZIPIII and KAN genes
are involved in more general patterning systems that evolved
early in or before land plant evolution (Floyd and Bowman
2007). Studies to assess the roles of both gene families in
early plant lineages would greatly improve our understanding
of the evolution of the plant developmental program.
Lamina outgrowth: YABBY family
The precise mechanism whereby lamina outgrowth is promoted in response to adaxial–abaxial juxtaposition is not
fully understood, but members of the YABBY gene family,
which encode protein with zinc-finger and helix-loop-helix domains, are known to play a key role in lamina outgrowth. The
Arabidopsis genome contains six YABBY genes [FILAMENTOUS
FLOWER (FIL), YABBY2 (YAB2), YAB3, YAB5, CRABS CLAW
(CRC) and INNER NO OUTER (INO)] (Bowman and Smyth
1999, Sawa et al. 1999, Siegfried et al. 1999, Villanueva et al.
1999), four of which (FIL, YAB2, YAB3 and YAB5) are expressed
in vegetative leaf primordia. Expression of the remaining two,
CRC and INO, is restricted to the floral organs. In Arabidopsis,
YABBY genes are expressed in the abaxial domain of lateral
organs and their overexpression results in the differentiation
of abaxial cell types on adaxial sides (Sawa et al. 1999, Siegfried
et al. 1999). Thus, YABBY genes were first proposed as an
abaxial determinant; however, subsequent studies point to
their primary roles in lamina outgrowth. In Arabidopsis, individual loss-of-function mutants of YABBY genes have no discernible phenotype in leaves; however, progressive loss of
YABBY function results in a dramatic loss of lamina expansion.
The fil yab3 double mutant develops narrow leaves with partial
loss of polar differentiation (Siegfried et al. 1999). Quadruple
mutants of the four vegetatively expressed YABBY genes (fil
yab235) shows more dramatic loss of lamina expansion, ranging
from fully radialized leaves to narrow leaves with some lamina
(Sarojam et al. 2010). Similarly, loss of YABBY activity by the
graminifolia (gram) mutation in A. majus also results in a reduction of leaf width, and further loss of YABBY activity by the
gram prolongata double mutation results in an almost complete loss of lamina development (Golz et al. 2004). In these
mutant leaves, polar differentiation is reduced and residual
lamina lacks marginal characters. Global and marker gene
expression analyses in yabby quadruple mutant leaves of
Arabidopsis demonstrate that adaxial–abaxial polarity is initially established but not maintained (Sarojam et al. 2010).
Thus, YABBY genes are not required for the initial establishment of leaf polarity, but rather are necessary for initiating
lamina outgrowth and maintenance of polarity. Consistently,
in many plant species, YABBY expression becomes localized to
the boundary between the adaxial and abaxial domains at the
leaf margins, where lamina outgrowth occurs (Eshed et al.
2004, Gleissberg et al. 2005, Juarez et al. 2004b, Tononi et al.
Plant Cell Physiol. 53(7): 1180–1194 (2012) doi:10.1093/pcp/pcs074 ! The Author 2012.
Adaxial–abaxial polarity and lamina outgrowth
2010). Interestingly, expression of YABBY genes is regulated
by polarity pathways, such as the KANADI, HD-ZIPIII and AS
pathways (Eshed et al. 2004, Garcia et al. 2006, Juarez et al.
2004b, Li et al. 2005, Siegfried et al. 1999). Furthermore, YABBY
genes are strongly expressed in the ectopic outgrowth formed
at the ectopic adaxial–abaxial juxtaposition seen in weak
polarity mutants (Eshed et al. 2004, Juarez et al. 2004b) and
YABBY gene activity is required for ectopic outgrowth in the
kan1 kan2 double mutant (Eshed et al. 2004). Therefore,
YABBY genes function to integrate polarity signals to promote
lamina outgrowth. Whereas most YABBY genes are expressed in
the abaxial domain of lateral organs in eudicots and in basal
angiosperms, some YABBY genes in Poaceae are expressed
adaxially (Juarez et al. 2004b), centrally (Ishikawa et al. 2009,
Yamaguchi et al. 2004, Yamaguchi et al. 2010) or throughout
lateral organs (Dai et al. 2007, Tanaka et al. 2012). These findings suggest that upstream pathways that regulate expression
patterns of YABBY genes are diverse among angiosperms.
YABBY genes are also required to repress genetic programs
of the SAM in developing leaves and to initiate maturation
programs of leaves. Class I KNOX genes are ectopically expressed in fil yab3 double mutant leaves (Kumaran et al.
2002). In transgenic plants that express a synthetic microRNA
designed to target FIL and YAB3, WUSCHEL (WUS) is expressed
at the adaxial tips in a shoot-like manner (Sarojam et al. 2010).
On the other hand, most CINCINNATA-class TCP genes, which
promote leaf maturation programs (Efroni et al. 2008, Nath
et al. 2003, Ori et al. 2007, Palatnik et al. 2003), are not activated in yabby quadruple mutant leaves (Sarojam et al. 2010).
Thus, YABBY genes are involved in the regulation of determinacy, dorsoventrality and lamina outgrowth, which distinguish
leaves from stems. Since the origin of the YABBY gene family is
coincident with the evolution of seed plant leaves, YABBY genes
may have been important in the origin and evolution of seed
plant leaves (Floyd and Bowman 2007, Floyd and Bowman
2010). Analysis of YABBY gene function in gymnosperms as
well as in diverse angiosperms may therefore reveal their ancestral roles and their involvement in the evolution of seed
plant leaves.
of leaf primordia in a pattern similar to that of NS genes
(Matsumoto and Okada 2001). The leaf phenotype of the prs
mutant is limited to the deletion of stipules, which are marginal
structures in the leaf base, with no reduction in leaf width as
seen in maize (Matsumoto and Okada 2001, Nardmann et al.
2004). Loss of the prs phenotype in leaves is due to genetic
redundancy with WOX1, which belongs to another subfamily
of WOX genes. In Arabidopsis, WOX1 is expressed along the
adaxial–abaxial juxtaposition, overlapping that of PRS at the
leaf margins (Nakata et al. 2012, Vandenbussche et al. 2009).
Although wox1 single mutants show no visible phenotype, the
prs wox1 double mutant causes severe defects in lamina outgrowth (Nakata et al. 2012, Vandenbussche et al. 2009). Thus,
PRS and WOX1 act redundantly to promote lateral lamina outgrowth. In some plant species, single loss-of-function of WOX1
subfamily genes, such as MAEWEST (MAW) in petunia
(Petunia hybrida), STENOFOLIA in Medicago truncatula and
LAM1 in Nicotiana sylvestris, result in a severe reduction in leaf
width (Tadege et al. 2011, Vandenbussche et al. 2009). In prs
wox1 or maw mutant leaves, adaxial–abaxial polarity is slightly
disturbed at the leaf margins, suggesting that these genes adjust
the balance between adaxial and abaxial domains at leaf margins (Nakata et al. 2012, Vandenbussche et al. 2009). On the
other hand, overall adaxial–abaxial polarity is not disrupted in
these mutant leaves (Tadege et al. 2011, Vandenbussche et al.
2009), indicating that WOX genes act downstream of the polarity pathway to promote lamina outgrowth. It is possible that
adaxial–abaxial polarity defines WOX gene expression along the
adaxial–abaxial boundary or at leaf margins, where WOX genes
promote lamina outgrowth. Indeed, Nakata et al. (2012)
demonstrated that the expression of PRS and WOX1 is under
the control of adaxial–abaxial polarity determinants. The expression of PRS and WOX1 is negatively regulated by KAN, and
is positively regulated by FIL. They also showed that PRS and
WOX1, in turn, restrict the expression of AS1 to the adaxial
domain. Further analysis of the regulatory mechanism of
WOX gene expression by polarity pathways and the relationship
between WOX and YABBY genes would reveal the precise
mechanism whereby lamina outgrowth is promoted in
response to adaxial–abaxial juxtaposition.
Lamina outgrowth: WOX family
Members of at least two subfamilies of WUS-related homeobox
(WOX) genes play an essential role in lamina outgrowth: one is
the PRESSED FLOWER (PRS)/WOX3 subfamily and the other is
the WOX1 subfamily. The PRS/WOX3 subfamily includes PRS of
Arabidopsis and NARROW SHEATH1 (NS1) and NS2 of maize
(Matsumoto and Okada 2001, Nardmann et al. 2004). In the
ns1 ns2 double mutant, leaf width is severely reduced as a result
of deletion of leaf lateral domains (Nardmann et al. 2004). NS1
and NS2 are expressed in the leaf margins and in the two lateral
foci in the SAM, where they recruit leaf founder cells, which give
rise to leaf lateral and marginal domains (Nardmann et al.
2004). In Arabidopsis, PRS is expressed in marginal domains
Lamina outgrowth: auxin
Auxin is a primary plant hormone. The differential distribution
of auxin, which is generated by biosynthesis and polar transport
within plant tissues, affects various aspects of plant growth and
development (Bowman and Floyd 2008, Vanneste and Friml
2009). During leaf development, an auxin maximum is first
formed at the tips of young leaf primordia, which is thought
to direct their distal growth (Benkova et al. 2003, Reinhardt
et al. 2003). Subsequently, the auxin, derived from the leaf
margins, is symmetrically distributed on either side of the midvein, where it is thought to facilitate blade outgrowth (Aloni
et al. 2003, Mattsson et al. 1999, Scanlon 2003, Zgurski et al.
Plant Cell Physiol. 53(7): 1180–1194 (2012) doi:10.1093/pcp/pcs074 ! The Author 2012.
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T. Yamaguchi et al.
2005). Several lines of evidence also suggest that auxin acts
downstream of polarity genes to promote lamina outgrowth.
In the kan1 kan2 kan4 triple mutant, ectopic lamina outgrowth
on the hypocotyls is associated with ectopic localization of an
auxin transporter PIN-FORMED1 (PIN1) protein (Izhaki and
Bowman 2007). Loss of YABBY activity greatly influences the
distribution of auxin and localization of PIN1 (Sarojam et al.
2010). Furthermore, in as1 and as2 mutants, asymmetric distribution of auxin is associated with asymmetrical lamina outgrowth (Zgurski et al. 2005).
Recent studies on YUCCA (YUC) genes revealed more direct
evidence that auxin plays a key role in the promotion of lamina
outgrowth and leaf margin formation, acting downstream of
the adaxial–abaxial polarity pathway. Members of the YUC
gene family encode flavin monooxygenases, which catalyze
the rate-limiting step in Trp-dependent auxin biosynthesis
and play important roles in local auxin biosynthesis (Zhao
et al. 2001). The Arabidopsis genome contains 11 YUC genes,
and removal of at least four genes (YUC1, YUC2, YUC4 and
YUC6) results in plants with narrow leaves and loss of leaf marginal characters (Cheng et al. 2006, 2007, Wang et al. 2011).
Expression of these genes is associated with leaf margins and
hydathodes during leaf development (Wang et al. 2011).
Interestingly, YUC genes are up-regulated in ectopic lamina
outgrowth, which is formed at the ectopic adaxial and abaxial
juxtaposition in kan1 kan2 and as2 rev double mutant leaves
(Eshed et al. 2001, Eshed et al. 2004, Fu et al. 2007). In addition, multiple loss of YUC activity in kan1 kan2 and as2 rev
double mutants also results in a loss of ectopic lamina outgrowth (Wang et al. 2011). In rice, loss of YUC activity by
constitutively wilted1/narrow leaf7 mutations also results in
the reduction of endogenous auxin content and a narrow leaf
phenotype (Fujino et al. 2008, Woo et al. 2007). Thus, YUC
genes are expressed in response to adaxial–abaxial juxtaposition, and local auxin synthesis by YUC is partly responsible
for lamina outgrowth and leaf margin development in
angiosperms.
Distinct regulation of adaxial–abaxial
patterning in stamens
The adaxial–abaxial patterning system has been co-opted to
generate complex organ morphologies. For example, the development of bi-keeled prophylls in Poaceae is associated with an
alteration in adaxial–abaxial polarity (Johnston et al. 2010).
Floral organs are thought to be modified leaves and, thus,
likely share common developmental programs. The morphology of stamens, however, is distinct from that of leaves. The
stamen consists of two morphologically distinct parts: a proximal filament and a distal anther. A typical anther consists of
two thecae and each theca contains a pair of microsporangia
(pollen sacs). On the other hand, the filament is a radially symmetrical structure and serves as a stalk (Goldberg et al. 1993).
Recently, Toriba et al. (2010) demonstrated that dynamic
1186
rearrangement of adaxial–abaxial polarity defines the basic
structures of stamens in rice. In an early stage of stamen development, adaxial–abaxial polarity is established as in leaf primordia: OsPHB3 (a PHB ortholog) is expressed in the adaxial domain
and OsETTIN1 (OsETT1: an ARF3/ETT ortholog) in the abaxial
domain. Subsequently, a dramatic rearrangement of adaxial–
abaxial polarity takes place both in anther and filament regions.
In the anther region, OsPHB3 is expressed in the lateral regions
of the primordium with concomitant loss of original adaxial
expression, while OsETT1 expression is seen in a region near
the meristem in addition to the original abaxial region. This
newly formed adaxial–abaxial polarity seems to correspond
to that of the theca primordium. Each of four boundaries between the adaxial and abaxial identities is thought to promote
outgrowths, which subsequently differentiate into pollen sacs.
In contrast, in the proximal filament region, the expression of
OsPHB3 disappears, suggesting that the filament is abaxialized
(Toriba et al. 2010). This idea is supported by observations of
abnormal stamen development in the rod-like lemma allele of
the SHOOTLESS2 locus (shl2-rol), in which adaxial–abaxial polarity is compromised by a weak mutation in the SHL gene,
which encodes an RNA-dependent RNA polymerase and functions in ta-siRNA production (Nagasaki et al. 2007, Toriba
et al. 2010). Thus, the establishment of adaxial–abaxial polarity
in the stamen is markedly different from that in leaves: independent establishment of polarity in the distal and proximal
regions followed by rearrangement of polarity in the anthers
and complete abaxialization of the filaments. Further studies on
the mechanism underlying the dynamic rearrangement of polarity during stamen development would reveal novel aspects
of the adaxial–abaxial polarity specification during organ
development.
Unifacial leaf development and evolution
Angiosperm leaves show remarkable morphological diversity,
thus making them an attractive subject for evolutionary developmental (evo-devo) studies (Piazza et al. 2005). Alterations to
adaxial–abaxial patterning mechanisms cause dramatic modifications in the leaf morphology and, thus, could be a major
driving force in the generation of diverse leaf forms (Gleissberg
et al. 2005, Johnston et al. 2010). For example, changes in ARP
expression and associated variation in adaxial–abaxial patterning in compound leaves is thought to be associated with the
evolution of peltate leaves and variation in leaflet placement
patterns (Kim et al. 2003, Luo et al. 2005). One of the most
interesting examples is the ‘unifacial leaf’, which is found in a
number of divergent monocot species (Kaplan 1975, Rudall and
Buzgo 2002, Yamaguchi and Tsukaya 2010, Yamaguchi et al.
2010). Monocot leaves usually consist of two morphologically
distinct domains along the proximal–distal axis: the proximal
leaf sheath and the distal leaf blade (Kaplan 1973, Rudall and
Buzgo 2002). In bifacial leaves, both the leaf sheath and
the leaf blade are dorsoventrally flattened and differentiate
Plant Cell Physiol. 53(7): 1180–1194 (2012) doi:10.1093/pcp/pcs074 ! The Author 2012.
Adaxial–abaxial polarity and lamina outgrowth
Fig. 3 Leaf blade structures and adaxial–abaxial polarity in bifacial and unifacial leaves. (A, B) Bifacial leaves in tulip (Tulipa gesneriana). (C, D)
Flattened unifacial leaves in German iris (Iris germanica). (E, F) Cylindrical unifacial leaves in Welsh onion (Allium fistulosum). (B, D, F) Schematic
diagrams of transverse sections through leaf blades. Positional relationships of leaves to the SAM are indicated by circles. (G, H) In situ localization
of JpPHB (G) and JpARF3a (H) transcripts in transverse sections of unifacial leaf primordia of J. prismatocarpus. (I, J) In situ localization of JpPHB
(I) and JpARF3a (J) transcripts in longitudinal sections through the SAM. (K) Adaxial and abaxial identities in a longitudinal section of a leaf
primordium in J. prismatocarpus. Arrow in (I) shows the adaxial expression of JpPHB only in the basal region of the leaf primordium, which
will differentiate into the leaf sheath. The outlined arrow in (L) shows the expression of JpARF3a throughout the distal region of the leaf
primordium, which will differentiate into the leaf blade. Ad, adaxial domain; Ab, abaxial domain; Bl, leaf blade; Sh, leaf sheath; Xy, xylem region.
Bars = 200 mm. Images are reproduced from Yamaguchi et al. 2010; Plant Cell 22: 2141–2155. Copyright ! American Society of Plant Biologists
(www.plantcell.org).
adaxial–abaxial polarity (Fig. 3A, B). In contrast, in unifacial
leaves, the leaf blade is bilaterally flattened (ensiform) or cylindrical (terete) and consists of only one surface type, the abaxial
side (Fig. 3C–F). The leaf sheath, on the other hand, has a
similar structure to that in bifacial leaves, with differentiation
of adaxial–abaxial polarity (Kaplan 1975, Yamaguchi and
Tsukaya 2010). The abaxialized property of the unifacial leaf
blade has been determined from histological characteristics.
For example, epidermal and mesophyll tissues have no dorsoventral differentiation, and vascular bundles are usually
arranged in a ring beneath the outer leaf surface with all the
xylem poles pointing to the center (Kaplan 1975, Yamaguchi
and Tsukaya 2010, Yamaguchi et al. 2010). On the other hand,
some plant species, such as Eucalyptus globulus or many C4
plants, develop equifacial leaves, in which the epidermal and
mesophyll tissues are identical on both the adaxial and abaxial
sides (Troll 1954). For example, mature leaves of E. globulus
expand vertically as a result of secondary torsion, so that
each side of the leaf receives equivalent light. Thus, there is
no ecological reason for their leaves to differentiate adaxial–
abaxial asymmetry. Equifacial leaves are a kind of bifacial leaf
and differ from unifacial leaves in that they have inherent adaxial–abaxial polarity, as evident from the vascular patterning
and developmental patterns of leaf primordia.
The mechanisms behind the development and evolution of
unifacial leaves have long been subjects of debate (for review,
see Kaplan 1975, Yamaguchi and Tsukaya 2010), but remain to
be studied at the molecular genetic level. Recently, we established a model research system of unifacial leaves focusing on
the genus Juncus (Juncaceae), which contains species with a
wide variety of leaf forms (Cutler 1969, Kirschner 2002a,
Kirschner 2002b, Yamaguchi and Tsukaya 2010) and is amenable to molecular genetic studies (Yamaguchi and Tsukaya
2010). Our recent study demonstrated that the unifacial leaf
blade in Juncus prismatocarpus is abaxialized at the gene expression level: an ARF3/ETT ortholog (JpARF3a) is expressed
throughout the outer region of the leaf blade, while a PHB
ortholog (JpPHB) is expressed only in the xylem region
(Fig. 3G, H) (Yamaguchi et al. 2010). Observations of developmental patterns of unifacial leaf primordia, together with gene
expression analysis, show that the distal region of the unifacial
leaf primordia is abaxialized from a very early stage, while the
adaxial identity is restricted to the basal domain (Fig. 3I–K)
(Yamaguchi et al. 2010). These results suggest that the genetic
program promoting abaxial identity works predominantly in
unifacial leaves and the distal region of leaf primordia may be
more sensitive to the abaxialization effect or a mechanism preventing adaxialization exists in the basal region. Alternatively,
the onset of an adaxial-promoting program is perhaps delayed
in unifacial leaves. It is possible that a genetic change or changes
in regulators of adaxial–abaxial polarity may have resulted in
unifacial leaf development. Further functional studies of each
Plant Cell Physiol. 53(7): 1180–1194 (2012) doi:10.1093/pcp/pcs074 ! The Author 2012.
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T. Yamaguchi et al.
regulator are expected to reveal the genetic mechanisms
behind leaf blade abaxialization in unifacial leaves as well as
their repeated evolution in monocots.
Lamina outgrowth in unifacial leaves
It is widely believed that lamina outgrowth is promoted at the
juxtaposition of adaxial and abaxial identities. Interestingly,
many unifacial-leaved species develop flattened leaf blades despite their leaf blades being abaxialized. This indicates that the
regulation of lamina outgrowth in unifacial leaf blades is not
totally identical to the adaxial–abaxial juxtaposition system in
bifacial leaves (Yamaguchi and Tsukaya 2010, Yamaguchi et al.
2010). Consistently, the leaf blade in unifacial leaves is flattened
by vigorous and directional cell proliferation towards the SAM
side, which is perpendicular to lamina flattening in bifacial
leaves (Yamaguchi et al. 2010). Comparative analysis using
two very closely related Juncus species, J. prismatocarpus with
flattened unifacial leaf blades and Juncus wallichianus with cylindrical unifacial leaf blades, revealed the DROOPING LEAF (DL)
ortholog as a strong candidate for flattening of the unifacial leaf
blade (Yamaguchi et al. 2010). DL is a member of the YABBY
gene family and belongs to the CRC/DL subfamily. In bifacial
leaves of rice, DL is expressed in the center of leaf primordia,
where DL regulates leaf midrib formation by promoting cell
proliferation towards the SAM side (Fig. 4A, B) (Yamaguchi
et al. 2004). In flattened unifacial leaves of J. prismatocarpus, DL
is strongly expressed in the median plane, along which vigorous
and directional cell proliferation occurs (Fig. 4C, D), whereas DL
is only weakly expressed in cylindrical unifacial leaves in
J. wallichianus (Fig. 4E, F). Genetic and expression analyses
using interspecific hybrids of the two species revealed that
the DL locus from J. prismatocarpus flattens the unifacial leaf
blade and expresses higher amounts of DL transcript than that
from J. wallichianus. DL plays a similar function at the cellular
level both in bifacial and unifacial leaf development, promoting
cell proliferation of leaf primordia towards the SAM. DL function in the promotion of cell proliferation may have been easily
co-opted to flatten abaxialized leaf blades during the evolution
of unifacial leaves (Fig. 4G). It is also of interest that YABBY
gene function is utilized to facilitate lamina outgrowth in unifacial leaves, although it has been suggested that CRC/DL subfamily genes are not involved in lamina outgrowth. They have a
conserved role in carpel development in angiosperms and
specific function in leaf midrib formation in monocots as well
as nectary formation in core eudicots (Bowman and Smyth
1999, Fourquin et al. 2005, Ishikawa et al. 2009, Lee et al.
2005, Nakayama et al. 2010, Yamada et al. 2011, Yamaguchi
et al. 2004). One possibility is that a more general role of
Fig. 4 Leaf midrib formation in bifacial leaves and lamina outgrowth in unifacial leaves by DL function. (A) A transverse section of a bifacial leaf
blade in rice. (B) In situ localization of DL transcripts in a transverse section of leaf primordia in rice, showing strong DL expression in the central
domain of leaf primordia. (C) A transverse section of a flattened leaf blade in J. prismatocarpus. (D) In situ localization of JpDL transcripts in a
transverse section of a P2 leaf blade, showing strong JpDL expression in the central domain of leaf primordia. (E) A transverse section of a
cylindrical leaf blade in J. wallichianus. (F) In situ localization of JwDL transcripts in a transverse section of a P2 leaf blade, showing weak JwDL
expression around only the central vascular bundle. Note that the internal air spaces in mature leaf blades as seen in (A, C, E) are formed by cell
death and the internal region of the young leaf primordium is occupied by dividing cells, as seen in (B, D, F). (G) Schematic of leaf midrib
formation in monocot bifacial leaves and laminar outgrowth in unifacial leaves by DL function. Bl, leaf blade; Cv, central large vascular bundle.
Bars = 200 mm. Images in (C–F) are reproduced from Yamaguchi et al. 2010; Plant Cell 22: 2141–2155. Copyright ! American Society of Plant
Biologists (www.plantcell.org).
1188
Plant Cell Physiol. 53(7): 1180–1194 (2012) doi:10.1093/pcp/pcs074 ! The Author 2012.
Adaxial–abaxial polarity and lamina outgrowth
YABBY genes is the promotion of directional outgrowth, used
in various contexts of plant development, such as lamina
outgrowth, carpel development, and midrib and nectary
formation.
Central–marginal polarity specification in
flattened leaves
In contrast to adaxial–abaxial polarity, how central–marginal
leaf polarity is specified remains largely unknown even in model
species, because of a lack of useful mutants and expression
markers. In bifacial leaves of monocots, DL and PRS/WOX3
homologs show a polar expression pattern along the central–
marginal axis. DL and its orthologs are expressed in the central
domain of leaf primordia (Ishikawa et al. 2009, Nakayama et al.
2010, Yamaguchi et al. 2004), while PRS homologs are
expressed at leaf margins (Nardmann et al. 2004, Nardmann
et al. 2007). Close observations of expression patterns of DL
and RPS homologs in unifacial leaves revealed that central–
marginal polarity is rearranged during lamina flattening. In
leaf primordia of J. prismatocarpus, JpDL is first expressed in
the central domain, which is similar to DL expression in bifacial
leaves. Subsequently, after flattening of the leaf blade, JpDL also
becomes expressed centrally to the flattened leaf blade
(Yamaguchi et al. 2010). In addition, no expression of a homolog of PRS/WOX3 (JpPRSb) is found before primordial flattening,
but is observed in both tips after flattening of the leaf blade
(Yamaguchi et al. 2010). These expression patterns of DL and
PRSb in flattened leaf blades suggest that central–marginal polarity partly differentiates along the flattened leaf shape. In contrast, no such expression has been observed in cylindrical
unifacial leaves of J. wallichianus or radialized leaves of radial
leaf mutants of J. prismatocarpus (Yamaguchi et al. 2010).
These observations indicate that central–marginal polarity
can somewhat autonomously differentiate, depending on the
flattened leaf shape. Expression of JpPRSb in margin-like domains of abaxialized leaf blades also indicates that the leaf
margin identity could be partly independent of adaxial–abaxial
juxtaposition. We hypothesize that a gradient of central–marginal polarity is formed by an as yet unidentified molecule,
which is distributed in a symmetrical pattern in the flattened
leaf blade, triggering the expression of DL and PRSb. The plant
hormone auxin serves as a gradient signal in multiple contexts
of plant development (Bowman and Floyd, 2008, Vanneste and
Friml 2009) and is therefore a potential candidate for this unidentified molecule. Alternatively, physical factors, such as tension, could also be considered as candidates that regulate
central–marginal polarity differentiation. Whether JpPRSb expression in margin-like regions acts to promote flattening of
unifacial leaf blades at later stages in addition to DL is also of
interest. Isolation of JpPRSb mutants from J. prismatocarpus
would help to reveal the role of this gene in flattening of the
unifacial leaf blade as well as the genetic mechanism underlying
leaf margin specification.
Conclusions and perspectives
Molecular genetic studies in recent decades have greatly expanded our understanding of the mechanism of adaxial–abaxial patterning and lamina flattening in angiosperms. These
studies have identified multiple regulators involved in leaf adaxial–abaxial patterning and lamina outgrowth, and have led to
the formation of a model in which antagonistic interactions
between adaxial and abaxial determinants establish polarity
with a subsequent promotion of lamina outgrowth. Further
clarification of the genetic and molecular interactions between
each regulator is an important next step towards a deeper
understanding of this mechanism. In particular, interactions
among lamina outgrowth regulators, such as YABBY, WOX
and auxin, remain obscure. It is also necessary to determine
the upstream regulatory mechanism that defines the expression or activities of lamina outgrowth regulators. How the adaxial–abaxial polarity is first established also remains unknown.
The identification and characterization of putative meristemderived positional information signals represent an important
challenge, with auxin or small RNAs thought to be candidates
for this signaling molecule (for review, see Husbands et al. 2009,
Kidner and Timmermans 2010). A recent report by Toyokura
et al. (2011) suggested that succinic semialdehyde or its close
derivatives could also be a signaling molecule.
Evo-devo studies on various leaf forms in angiosperms, such
as unifacial and compound leaves, suggest that alteration to the
adaxial–abaxial patterning system could be a major driving
force in the generation of diverse leaf forms. Investigation of
adaxial–abaxial polarity in leaves with a derived structure such
as peltate leaves found in many plant species (Gleissberg et al.
2005) or pitcher leaves found in some carnivorous plants
(Franck 1976) is also expected to deepen our understanding
of the evolutionary developmental mechanism of these leaves.
Another interesting example is the evolution of leaf-like stems
that exhibit flattened and dorsoventral structures. For example,
the genus Asparagus (Asparagaceae) has leaf-like organs called
cladodes in the axil of scale leaves (Cooney-Sovetts and Sattler
1986). A recent report by Nakayama et al. (2012) demonstrated that cladodes may have evolved by co-opting leaf developmental programs in axillary shoots. The evolution of
unusual shoot morphology in the subfamily Podostemoideae
(Podostemaceae) is also accompanied by the addition of a leaf
development program in shoots (Katayama et al. 2010). It is
possible that a master regulator that specifies leaf identity is
ectopically expressed in shoots of these plants. Thus, further
studies on these plants would give a unique chance to study the
mechanism of leaf identity specification. An important challenge in evo-devo studies is to identify genetic alterations responsible for leaf form evolution and to elucidate the genetic
basis underlying the morphological evolution.
Results from molecular genetics in angiosperms, together
with expression studies in non-seed plants, are beginning to
shed light on the origin and evolutionary processes of leaves.
Many regulators involved in adaxial–abaxial patterning were
Plant Cell Physiol. 53(7): 1180–1194 (2012) doi:10.1093/pcp/pcs074 ! The Author 2012.
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T. Yamaguchi et al.
present very early in land plant evolution, but their actual functions in development remains largely speculative (Floyd and
Bowman 2007). Elucidation of the ancestral roles of these regulators through functional analyses of early land plants is an
important challenge in revealing the evolutionary steps of
leaves.
Funding
This work was supported by a Grant-in-Aid for Young Scientists
(B) to T.Y. from the Ministry of Education, Culture, Sports,
Science and Technology (MEXT), Japan; a grant from the
Sumitomo Foundation to T.Y.; a Grant-in-Aid for Creative
Scientific Research to H.T. from the Japan Society for the
Promotion of Science (JSPS); a Grant-in-Aid for Scientific
Research on Priority Areas to H.T. from MEXT; and
a Grant-in-Aid for Young Scientists (Start-up) to A.N. from JSPS.
Acknowledgments
We thank Dr. Makoto Matsuoka, former Editor-in-Chief, and
Dr. Makoto Hayashi, Editor, for their kind invitation to publish
this review article in Plant and Cell Physiology.
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