Leaf adaxial abaxial polarity specification and lamina outgrowth: evolution and development

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1 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, Japan *Corresponding author: , Fax/Tel, (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): (2012) doi: /pcp/pcs074, available online at The Author Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please journals.permissions@oup.com 1180 Plant Cell Physiol. 53(7): (2012) doi: /pcp/pcs074! The Author 2012.

2 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. 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 A Lamina outgrowth B Ab Ad Adaxial Abaxial Wild-type leaf Ad Ab Separation from the SAM Signal 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 Abaxialized radial leaf Lamina outgrowth Abaxialized 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: Copyright! The Company of Biologists. 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): (2012) doi: /pcp/pcs074! The Author

3 T. Yamaguchi et al. 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 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) 1182 Plant Cell Physiol. 53(7): (2012) doi: /pcp/pcs074! The Author 2012.

4 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 HD-ZIPIII AS1 ARP TAS3 tasir-arf 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 Adaxial domain Lamina outgrowth PRS WOX1 YABBY mir165/166 KAN ARF3 ARF4 Abaxial domain 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. Auxin Plant Cell Physiol. 53(7): (2012) doi: /pcp/pcs074! The Author

5 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 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): (2012) doi: /pcp/pcs074! The Author 2012.

6 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. 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 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: 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): (2012) doi: /pcp/pcs074! The Author

7 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 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 1186 Plant Cell Physiol. 53(7): (2012) doi: /pcp/pcs074! The Author 2012.

8 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: Copyright! American Society of Plant Biologists ( 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 C 4 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): (2012) doi: /pcp/pcs074! The Author

9 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: Copyright! American Society of Plant Biologists ( Plant Cell Physiol. 53(7): (2012) doi: /pcp/pcs074! The Author 2012.

10 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): (2012) doi: /pcp/pcs074! The Author