Adaxial Domain Research Articles

STRUCTURAL AND REGULATORY ASPECTS OF MORPHOGENESIS IN <i>EQUISETUM</i> <i>SYLVATICUM</i> AND <i>EQUISETUM</i> <i>FLUVIATILE</i> AND THE ISSUE OF HOMOLOGY OF LEAVES OF HORSETAILS AND OTHER FERNS

The structure of the shoot apical meristem (SAM), ultrastructure of its cells and the origin of primordia of organs in the SAM of horsetails Equisetum sylvaticum L. and E. fluviatile L. were studied. A relatively low degree of vacuolation and structural homogeneity of the cells that compose the surface initials zone (SI), the presence of single starch grains in their plastids and single lipid droplets in the cytoplasm of the SI cells were revealed. These features are more similar to those in the monoplex SAM of lycophytes than to those in leptosporangiate ferns. It is shown that initiation of leaves in E. sylvaticum and E. fluviatile via emergence of the leaf apical initials (LAIs) in the surface initials zone is similar to that of other plants with monoplex SAM; both ferns and lycophytes. The main peculiarity of organogenesis in horsetails is the origin of the leaf whorl as a single structure; each whorl develops from the triplet of the shoot apical initial merophytes. The inner cells of the emerging leaf whorl that belong to the single triplet of merophytes differentiate into the intercalary meristem. Each phytomer of E. sylvaticum and E. fluviatile is composed of the whorl of congenitally fused leaves and a whorl of buds that emerge in the axils of leaf bases located between the blades and thus alternate with leaves. Each bud bears a single root primordium; bud primordia develop on aerial shoots while root primordia – on the underground shoots. Termination of the leaf apical meristem functioning, that consequences from the vacuolation of the LAI, leads to the absence of the marginal meristem and the vascular tissues in the leaf blade. Proximodistal development of the leaf whorl and origin of buds results from meristematic activity of the peripheral part of the intercalary meristem. The search for the homologues of genes encoding known for angiosperms regulators of the development of adaxial (C3HDZ and ARP) and abaxial (YABBY and KANADI) leaf domains in available transcriptomes of horsetails and matching of the found homologues with that from the genomes of mosses, ferns and gymnosperms was undertaken in the context of assessment of presumptive regulatory similarities or differences of leaves of horsetails and other ferns in the context of leaf homology in the two groups. MEME analysis has shown that R2-R3 MYB proteins from transciptomes of horsetails are not ARP homologues. Thus it has been revealed that horsetail have single adaxial domain regulator (C3HDZ) and single abaxial domain regulator (KANADI), as other ferns. This finding suggests the probable loss of other regulators of the adaxial (ARP) and abaxial (YABBY) domains in the common ancestor of Polypodiophyta. Phylogenetic analysis of the identified homologues of genes that encode WOX proteins suggest that the T3 clade, that includes regulators of marginal (WOX3) and plate (WOX1) leaf meristems, originated in a common ancestor of Polypodiophyta, also indicating similarities in the molecular genetic regulation of the leaves in horsetails and other ferns.

Insight into the formation of trumpet and needle-type leaf in Ginkgo biloba L. mutant.

The leaf type of a plant determines its photosynthetic efficiency and adaptation to the environment. The normal leaves of modern Ginkgo biloba, which is known as a "living fossil" in gymnosperm, evolved from needle-like to fan-shaped with obvious dichotomous venation. However, a newly discovered Ginkgo variety "SongZhen" have different leaf types on a tree, including needle-, trumpet-, strip-, and deeply split fan-shaped leaves. In order to explore the mechanism in forming these leaf types, the microscopy of different leaf types and transcriptome analysis of apical buds of branches with normal or abnormal leaves were performed. We found that the normal leaf was in an intact and unfolded fan shape, and the abnormal leaf was basically split into two parts from the petiole, and each exhibited different extent of variation. The needle-type leaves were the extreme, having no obvious palisade and spongy tissues, and the phloem cells were scattered and surrounded by xylem cells, while the trumpet-type leaves with normal vascular bundles curled inward to form a loop from the abaxial to adaxial side. The other type of leaves had the characteristics among needle-type, trumpet-type, or normal leaves. The transcriptome analysis and quantitative PCR showed that the genes related to abaxial domain were highly expressed, while the adaxial domain promoting genes were decreasingly expressed in abnormal-type leaf (ANL) buds and abnormal leaves, which might lead to the obvious abaxialized leaves of "SongZhen." In addition, the low expression of genes related to leaf boundary development in ANL buds indicated that single- or double-needle (trumpet) leaves might also be due to the leaf tissue fusion. This study provides an insight into the mechanism of the development of the abnormal leaves in "SongZhen" and lays a foundation for investigating the molecular mechanism of the leaf development in gymnosperms.

Differential growth dynamics control aerial organ geometry

How gene activities and biomechanics together direct organ shapes is poorly understood. Plant leaf and floral organs develop from highly similar initial structures and share similar gene expression patterns, yet they gain drastically different shapes later-flat and bilateral leaf primordia and radially symmetric floral primordia, respectively. We analyzed cellular growth patterns and gene expression in young leaves and flowers of Arabidopsis thaliana and found significant differences in cell growth rates, which correlate with convergence sites of phytohormone auxin that require polar auxin transport. In leaf primordia, the PRESSED-FLOWER-expressing middle domain grows faster than adjacent adaxial domain and coincides with auxin convergence. In contrast, in floral primordia, the LEAFY-expressing domain shows accelerated growth rates and pronounced auxin convergence. This distinct cell growth dynamics between leaf and flower requires changes in levels of cell-wall pectin de-methyl-esterification and mechanical properties of the cell wall. Data-driven computer model simulations at organ and cellular levels demonstrate that growth differences are central to obtaining distinct organ shape, corroborating in planta observations. Together, our study provides a mechanistic basis for the establishment of early aerial organ symmetries through local modulation of differential growth patterns with auxin and biomechanics.

Leaf dorsoventrality candidate gene CpARF4 has conserved expression pattern but divergent tasiR-ARF regulation in the water fern Ceratopteris pteridoides.

Leaves are traditionally classified into microphylls and megaphylls, and recently have been regarded as independently originating in lycophytes, ferns, and seed plants. The developmental genetics of leaf dorsoventrality, a synapomorphy in vascular plants, has been extensively studied in flowering plants. AUXIN RESPONSE FACTOR4 (ARF4) genes are key to leaf abaxial identity in flowering plants, but whether they exist in ferns is still an open question. ARF4 genes from Ceratopteris pteridoides, Cyrtomium guizhouense, and Parathelypteris nipponica were mined from transcriptomes and investigated in terms of evolutionary phylogeny and sequence motifs, with a focus on the tasiR-ARF binding site. In situ hybridization was used to localize expression of CpARF4 in Ceratopteris pteridoides. 5'RNA ligase-mediated-RACE was employed to verify whether CpARF4 transcripts were sliced by tasiR-ARF. ARF4 genes exist in ferns, and this lineage originates from a gene duplication in the common ancestor of ferns and seed plants. ARF4 genes are of a single copy in the ferns studied here, and they contain divergent and, at most, one tasiR-ARF binding site. CpARF4 is expressed in the abaxial but not the adaxial domain of leaf primordia at various developmental stages. Transcript slicing guided by tasiR-ARF is active in C. pteridoides, but CpARF4 probably has not been affected by it. Fern ARF4 genes differ in copy number and tasiR-ARF regulation relative to flowering plants, though they can be similarly expressed in the abaxial domain of leaves, revealing a key role for ARF4 genes in the evolution of leaf dorsoventrality of vascular plants.

Interaction between Floral Merism and Symmetry: Evidence from Fasciated Mutant of Lupinus angustifolius L. (Leguminosae)

A significant diversity of flower structure in angiosperms is defined by few parameters, among which one may list floral merism and symmetry as the most important. However, observation of normal variation in different taxa indicates that high floral merism (more than six organs) is very rarely associated with a monosymmetry. Precise mechanisms underlying this tendency, as well as the mode of interaction between regulatory pathways of floral symmetry and merism, remain unidentified. In this work, we observed the floral ontogeny in normal plants of Lupinus angustifolius L. (Leguminosae), described the floral structure and its variations in fasciated mutant. Our results suggest that monosymmetry is regulated independently from the floral merism, so three types of petals differentiate properly even in flowers with an anomalously high number of organs. However, the adaxial floral domain is the most stable floral part, which is the least susceptible to the fluctuations of merism. This means that some genes responsible for the development of the adaxial flower domain may additionally stabilize it and hence prevent fluctuations of merism in the adaxial domain. The monosymmetry in Leguminosae is a character connected with pollination by insects and requires a precise interaction between all floral parts. That is why fluctuations, which impair this interaction and symmetry, may decrease the pollination efficiency.

Morphogenesis of flattened unifacial leaves in Juncus prismatocarpus (Juncaceae).

To reveal the mode of morphogenesis of flattened unifacial leaves, we analysed the cell division direction and distribution on the leaf blade of Juncus prismatocarpus. Using the pulse-chase 5-ethynyl-2'-deoxyuridine method, we quantified and mapped the cell division direction on the leaf blade of J.prismatocarpus and compared the distribution of thickening cell divisions with the expression pattern of DROOPING LEAF (DL), a key gene involved in leaf blade thickening. Thickening cell divisions were the most abundant (>45%) among all cell division directions on the leaf blade of J.prismatocarpus from the early plastochron2 stage through the plastochron3 stage. Mapping of cell divisions indicated that cell divisions in a particular direction were not restricted to a particular domain but were distributed diffusely throughout the entire cross-sectional area of the leaf blade. Gradient analysis indicated that the distribution of thickening cell divisions of the adaxial domain was denser than that of the abaxial domain. Contrary to the prolonged and diffuse distribution of thickening cell divisions, DL expression was transient and restricted in a narrow band. Our results suggest that a diffuse 'thickening meristem' plays the key role in the development of flattened unifacial leaves.

Auxin polar transport flanking incipient primordium initiates leaf adaxial-abaxial polarity patterning.

The leaves of most higher plants are polar along their adaxial-abaxial axis, and the development of the adaxial domain (upper side) and the abaxial domain (lower side) makes the leaf a highly efficient photosynthetic organ. It has been proposed that a hypothetical signal transported from the shoot apical meristem (SAM) to the incipient leaf primordium, or conversely, the plant hormone auxin transported from the leaf primordium to the SAM, initiates leaf adaxial-abaxial patterning. This hypothetical signal has been referred to as the Sussex signal, because the research of Ian Sussex published in 1951 was the first to imply its existence. Recent results, however, have shown that auxin polar transport flanking the incipient leaf primordium, but not the Sussex signal, is the key to initiate leaf polarity. Here, we review the new findings and integrate them with other recently published results in the field of leaf development, mainly focusing on the early steps of leaf polarity establishment.

A genetic link between epigenetic repressor AS1–AS2 and DNA replication factors in establishment of adaxial–abaxial leaf polarity of <i>Arabidopsis</i>

Balanced development of adaxial and abaxial domains in leaf primordia is critical for the formation of flat symmetric leaf lamina. Arabidopsis ASYMMETRIC LEAVES1 (AS1) and AS2 proteins form a complex (AS1-AS2), which acts as key regulators for the adaxial development by the direct repression of expression of the abaxial gene ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3). Many modifier mutations have been identified, which enhance the defect of as1 and as2 mutations to generate abaxialized filamentous leaves without adaxial traits, suggesting that the development of the adaxial domain is achieved by cooperative repression by AS1-AS2 and the wild-type proteins corresponding to the modifiers. Mutations of several genes for DNA replication-related chromatin remodeling factors such as Chromatin Assembly Factor-1 (CAF-1) have been also identified as modifiers. It is still unknown, however, whether mutations in genes involved in DNA replication themselves might act as modifiers. Here we report that as1 and as2 mutants grown in the presence of hydroxyurea, a known inhibitor of DNA replication, form abaxialized filamentous leaves in a concentration-dependent manner. We further show that a mutation of the INCURVATA2 (ICU2) gene, which encodes the putative catalytic subunit of DNA polymerase α, and a mutation of the Replication Factor C Subunit3 (RFC3) gene, which encodes a protein used in replication as a clamp loader, act as modifiers. In addition, as2-1 icu2-1 double mutants showed increased mRNA levels of the genes for leaf abaxialization. These results suggest a tight link between DNA replication and the function of AS1-AS2 in the development of flat leaves.

Model for the role of auxin polar transport in patterning of the leaf adaxial-abaxial axis.

Leaf adaxial-abaxial polarity refers to the two leaf faces, which have different types of cells performing distinct biological functions. In 1951, Ian Sussex reported that when an incipient leaf primordium was surgically isolated by an incision across the vegetative shoot apical meristem (SAM), a radialized structure without an adaxial domain would form. This led to the proposal that a signal, now called the Sussex signal, is transported from the SAM to emerging primordia to direct leaf adaxial-abaxial patterning. It was recently proposed that instead of the Sussex signal, polar transport of the plant hormone auxin is critical in leaf polarity formation. However, how auxin polar transport functions in the process is unknown. Through live imaging, we established a profile of auxin polar transport in and around young leaf primordia. Here we show that auxin polar transport in lateral regions of an incipient primordium forms auxin convergence points. We demonstrated that blocking auxin polar transport in the lateral regions of the incipient primordium by incisions abolished the auxin convergence points and caused abaxialized leaves to form. The lateral incisions also blocked the formation of leaf middle domain and margins and disrupted expression of the middle domain/margin-associated marker gene WUSCHEL-RELATED HOMEOBOX 1 (SlWOX1). Based on these results we propose that the auxin convergence points are required for the formation of leaf middle domain and margins, and the functional middle domain and margins ensure leaf adaxial-abaxial polarity. How middle domain and margins function in the process is discussed.

A genetic link between epigenetic repressor AS1-AS2 and a putative small subunit processome in leaf polarity establishment of Arabidopsis.

ABSTRACTAlthough the DEAD-box RNA helicase family is ubiquitous in eukaryotes, its developmental role remains unelucidated. Here, we report that cooperative action between the Arabidopsis nucleolar protein RH10, an ortholog of human DEAD-box RNA helicase DDX47, and the epigenetic repressor complex of ASYMMETRIC-LEAVES1 (AS1) and AS2 (AS1-AS2) is critical to repress abaxial (ventral) genes ETT/ARF3 and ARF4, which leads to adaxial (dorsal) development in leaf primordia at shoot apices. Double mutations of rh10-1 and as2 (or as1) synergistically up-regulated the abaxial genes, which generated abaxialized filamentous leaves with loss of the adaxial domain. DDX47 is part of the small subunit processome (SSUP) that mediates rRNA biogenesis. In rh10-1 we found various defects in SSUP-related events, such as: accumulation of 35S/33S rRNA precursors; reduction in the 18S/25S ratio; and nucleolar hypertrophy. Double mutants of as2 with mutations of genes that encode other candidate SSUP-related components such as nucleolin and putative rRNA methyltransferase exhibited similar synergistic defects caused by up-regulation of ETT/ARF3 and ARF4. These results suggest a tight link between putative SSUP and AS1-AS2 in repression of the abaxial-determining genes for cell fate decisions for adaxial development.

ASYMMETRIC LEAVES2-LIKE38, one member of AS2/LOB gene family, involves in regulating ab-adaxial patterning in Arabidopsis lateral organs

ASYMMERTIC LEAVES1 (AS1) and AS2 in Arabidopsis are essential in determining leaf cell fates of adaxial axis. Here, we report ASYMMERTIC LEAVES2-LIKE38 (ASL38/LBD41), is an important gene of the LATERAL ORGAN BOUNDARY DOMAIN family. ASL38/LBD41 was detected in the adaxial and internal domain between the ab-adaxial domains of leaves. For explaining ASL38/LBD41 role in Arabidopsis development, a construct of the sense-expressing ASL38/LBD41 was transformed to Arabidopsis (Col-0); thus gained both overexpressing and cosuppressing 35S:ASL38/LBD41 plants. Rosette leaves of these overexpressing plants showed narrow (Class I) to radial symmetric needle-like patterns (Class II), compared with those of wild-type control, exhibiting adaxilized defect. However, rosette leaves of these cosuppressing plants showed narrow (Class I) to radial symmetric needle-like patterns (Class II), in contrast to those of wild-type (Col-0), exhibiting abaxilized defect. Furthermore, phenotypes observed in asl38-1 mutants suggest a redundant function for ASL38/LBD41 in boosting adaxial and/or inhibiting cell fate in abaxial axis. Together, our data suggest that ASL38/LBD41 might play a role of specialization in adaxial cell fate in Arabidopsis lateral organs.

Meta-Analysis of Arabidopsis KANADI1 Direct Target Genes Identifies a Basic Growth-Promoting Module Acting Upstream of Hormonal Signaling Pathways.

An intricate network of antagonistically acting transcription factors mediates the formation of a flat leaf lamina of Arabidopsis (Arabidopsis thaliana) plants. In this context, members of the class III homeodomain leucine zipper (HD-ZIPIII) transcription factor family specify the adaxial domain (future upper side) of the leaf, while antagonistically acting KANADI transcription factors determine the abaxial domain (future lower side). Here, we used a messenger RNA sequencing approach to identify genes regulated by KANADI1 (KAN1) and subsequently performed a meta-analysis combining our data sets with published genome-wide data sets. Our analysis revealed that KAN1 acts upstream of several genes encoding auxin biosynthetic enzymes. When exposed to shade, we found three YUCCA genes, YUC2, YUC5, and YUC8, to be transcriptionally up-regulated, which correlates with an increase in the levels of free auxin. When ectopically expressed, KAN1 is able to transcriptionally repress these three YUC genes and thereby block shade-induced auxin biosynthesis. Consequently, KAN1 is able to strongly suppress shade-avoidance responses. Taken together, we hypothesize that HD-ZIPIII/KAN form the basis of a basic growth-promoting module. Hypocotyl extension in the shade and outgrowth of new leaves both involve auxin synthesis and signaling, which are under the direct control of HD-ZIPIII/KAN.

Oriented cell division shapes carnivorous pitcher leaves of Sarracenia purpurea.

Complex morphology is an evolutionary outcome of phenotypic diversification. In some carnivorous plants, the ancestral planar leaf has been modified to form a pitcher shape. However, how leaf development was altered during evolution remains unknown. Here we show that the pitcher leaves of Sarracenia purpurea develop through cell division patterns of adaxial tissues that are distinct from those in bifacial and peltate leaves, subsequent to standard expression of adaxial and abaxial marker genes. Differences in the orientation of cell divisions in the adaxial domain cause bifacial growth in the distal region and adaxial ridge protrusion in the middle region. These different growth patterns establish pitcher morphology. A computer simulation suggests that the cell division plane is critical for the pitcher morphogenesis. Our results imply that tissue-specific changes in the orientation of cell division underlie the development of a morphologically complex leaf.

Spatiotemporal sequestration of miR165/166 by Arabidopsis Argonaute10 promotes shoot apical meristem maintenance.

Arabidopsis Argonaute10 (AGO10) specifically sequesters miR165 and miR166 and antagonizes their activity, thus regulating shoot apical meristem (SAM) development. However, where and when this sequestration acts is currently unclear. We show here that AGO10 represses miR165/166 activity in the embryo proper during early embryogenesis, through the apical and central regions of mature embryos, and eventually in the entire adaxial domain and vasculature of the cotyledons and leaf primordia. These locations are essentially identical to regions expressing PHABULOSA and REVOLUTA, mRNA targets of miR165/166. The Arabidopsis genome contains nine MIR165/166 genes. Sequestration of miR165/166 by the MIR165b, MIR166a, MIR166b, and MIR166g promoters efficiently rescues the SAM defect in ago10 mutants. Comparison of the expression patterns of AGO10 and the four MIR165/166 members suggests that AGO10 quenches the non-cell-autonomous activity of any miR165/166 that moves into AGO10-expressing niches. Thus, this study provides insight into how the spatiotemporal regulation of AGO10-miR165/166 activity affects SAM development.

Quantitative control of ASYMMETRIC LEAVES2 expression is critical for leaf axial patterning in Arabidopsis

ASYMMETRIC LEAVES2 (AS2) is one of the key genes required for specifying leaf adaxial identity during leaf adaxial–abaxial polarity establishment. Previous data have shown that, in leaf development, AS2 is directly repressed by an abaxially located transcription factor KANADI1 (KAN1), so that the AS2 transcripts are restricted only in the adaxial leaf domain. It is shown here that, different from the spatial repression by KAN1, the quantitative repression of AS2 in the adaxial domain is also critical for ensuring normal leaf pattern formation. By analysing two gain-of-function as2 mutants, as2-5D and isoginchaku-2D (iso-2D), it is shown that the similar AS2-over-expressed phenotypes of these mutants reflect two different kinds of AS2 misexpression patterns. While as2-5D causes disruption of a KAN1-binding site at the AS2 promoter leading to derepression of AS2 in the abaxial side but without changing its expression level of a leaf, iso-2D results in over-expression of AS2 but without altering its adaxial expression pattern. In addition, it was found that, in iso-2D, levels of histone H3 lysine 27 trimethylation (H3K27me3) and H3K4me3 at the AS2 locus are significantly reduced and increased, respectively, compared with those in the wild type and as2-5D. These results suggest that during leaf patterning, quantitative control of the AS2 expression level might involve epigenetic regulations.

Dual regulation of ETTIN (ARF3) gene expression by AS1-AS2, which maintains the DNA methylation level, is involved in stabilization of leaf adaxial-abaxial partitioning in Arabidopsis

Leaf primordia are generated at the periphery of the shoot apex, developing into flat symmetric organs with adaxial-abaxial polarity, in which the indeterminate state is repressed. Despite the crucial role of the ASYMMETRIC LEAVES1 (AS1)-AS2 nuclear-protein complex in leaf adaxial-abaxial polarity specification, information on mechanisms controlling their downstream genes has remained elusive. We systematically analyzed transcripts by microarray and chromatin immunoprecipitation assays and performed genetic rescue of as1 and as2 phenotypic abnormalities, which identified a new target gene, ETTIN (ETT)/AUXIN RESPONSE FACTOR3 (ARF3), which encodes an abaxial factor acting downstream of the AS1-AS2 complex. While the AS1-AS2 complex represses ETT by direct binding of AS1 to the ETT promoter, it also indirectly activates miR390- and RDR6-dependent post-transcriptional gene silencing to negatively regulate both ETT and ARF4 activities. Furthermore, AS1-AS2 maintains the status of DNA methylation in the ETT coding region. In agreement, filamentous leaves formed in as1 and as2 plants treated with a DNA methylation inhibitor were rescued by loss of ETT and ARF4 activities. We suggest that negative transcriptional, post-transcriptional and epigenetic regulation of the ARFs by AS1-AS2 is important for stabilizing early leaf partitioning into abaxial and adaxial domains.

A role for PHANTASTICA in medio-lateral regulation of adaxial domain development in tomato and tobacco leaves

A role for PHANTASTICA in medio-lateral regulation of adaxial domain development in tomato and tobacco leaves

ASYMMETRIC-LEAVES2 and an ortholog of eukaryotic NudC domain proteins repress expression of AUXIN-RESPONSE-FACTOR and class 1 KNOX homeobox genes for development of flat symmetric leaves in Arabidopsis

SummaryLeaf primordia form around the shoot apical meristem, which consists of indeterminate stem cells. Upon initiation of leaf development, adaxial-abaxial patterning is crucial for appropriate lateral expansion, via cellular proliferation, and the formation of flat symmetric leaves. Many genes that specify such patterning have been identified, but regulation by upstream factors of the expression of relevant effector genes remains poorly understood. In Arabidopsis thaliana, ASYMMETRIC LEAVES2 (AS2) and AS1 play important roles in repressing transcription of class 1 KNOTTED1-like homeobox (KNOX) genes and leaf abaxial-determinant effector genes. We report here a mutation, designated enhancer of asymmetric leaves2 and asymmetric leaves1 (eal), that is associated with efficient generation of abaxialized filamentous leaves on the as2 or as1 background. Levels of transcripts of many abaxial-determinant genes, including ETTIN (ETT)/AUXIN RESPONSE FACTOR3 (ARF3), and all four class 1 KNOX genes were markedly elevated in as2 eal shoot apices. Rudimentary patterning in as2 eal leaves was suppressed by the ett mutation. EAL encodes BOBBER1 (BOB1), an Arabidopsis ortholog of eukaryotic NudC domain proteins. BOB1 was expressed in plant tissues with division potential and bob1 mutations resulted in lowered levels of transcripts of some cell-cycle genes and decreased rates of cell division in shoot and root apices. Coordinated cellular proliferation, supported by BOB1, and repression of all class 1 KNOX genes, ETT/ARF3 by AS2 (AS1) and BOB1 might be critical for repression of the indeterminate state and of aberrant abaxialization in the presumptive adaxial domain of leaf primordia, which might ensure the formation of flat symmetric leaves.

A role for PHANTASTICA in medio-lateral regulation of adaxial domain development in tomato and tobacco leaves

Diverse leaf forms in nature can be categorized into two groups: simple and compound. A simple leaf has a single blade unit, whilst a compound leaf is dissected into leaflets. For both simple and compound leaves, a MYB domain transcription factor PHANTASTICA (PHAN) plays an important role in establishing the adaxial domain in the leaf. Absence of PHAN in arabidopsis and antirrhinum leaves supresses blade development, and in tomato suppresses leaflet development. However, in the rachis and petiole regions of tomato leaves where PHAN and the adaxial domain coexist, it has been unclear why leaf blade and leaflets are not formed. We hypothesized that PHAN regulates the medio-lateral extent of the adaxial domain, thereby determining compound leaf architecture. To test this hypothesis, we generated and analysed transgenic tomato plants expressing tomato PHAN (SlPHAN) under the Cauliflower mosaic virus (CaMV) 35S promoter in both sense and antisense orientations, and tobacco plants that over-express tomato SlPHAN. Modulations in SlPHAN resulted in a variety of leaf morphologies such as simple, ternate and compound in either a peltate or non-peltate arrangement. Measurements of the extent of the adaxial domain along the wild-type tomato leaf axis showed that the adaxial domain is narrowed in the rachis and petiole in comparison with regions where laminar tissue arises. In antiSlPHAN transgenic leaves, no blade or leaflet was formed where the adaxial domain was medio-laterally narrowed, and KNOX gene expression was correlatively upregulated. CaMV35S::SlPHAN expression led to widening of the adaxial domain and ectopic blade outgrowth in the rachis of tomato and in the petiole of tobacco. Taken together, these results suggest that SlPHAN plays a role in medio-lateral extension of the adaxial domain and contributes to final leaf morphology in tomato. This study provides a novel insight into leaf architecture in tomato and highlights how changes in the expression domain of a master regulator gene such as SlPHAN can be translated into diverse final leaf morphologies.

ASYMMETRIC LEAVES2 and Elongator, a Histone Acetyltransferase Complex, Mediate the Establishment of Polarity in Leaves of Arabidopsis thaliana

Leaf primordia are generated around the shoot apical meristem. Mutation of the ASYMMETRIC LEAVES2 (AS2) gene of Arabidopsis thaliana results in defects in repression of the meristematic and indeterminate state, establishment of adaxial-abaxial polarity and left-right symmetry in leaves. AS2 represses transcription of meristem-specific class 1 KNOX homeobox genes and of the abaxial-determinant genes ETTIN/ARF3, KANADI2 and YABBY5. To clarify the role of AS2 in the establishment of leaf polarity, we isolated mutations that enhanced the polarity defects associated with as2. We describe here the enhancer-of-asymmetric-leaves-two1 (east1) mutation, which caused the formation of filamentous leaves with abaxialized epidermis on the as2-1 background. Levels of transcripts of class 1 KNOX and abaxial-determinant genes were markedly higher in as2-1 east1-1 mutant plants than in the wild-type and corresponding single-mutant plants. EAST1 encodes the histone acetyltransferase ELONGATA3 (ELO3), a component of the Elongator complex. Genetic analysis, using mutations in genes involved in the biogenesis of a trans-acting small interfering RNA (ta-siRNA), revealed that ELO3 mediated establishment of leaf polarity independently of AS2 and the ta-siRNA-related pathway. Treatment with an inhibitor of histone deacetylases (HDACs) caused additive polarity defects in as2-1 east1-1 mutant plants, suggesting the operation of an ELO3 pathway, independent of the HDAC pathway, in the determination of polarity. We propose that multiple pathways play important roles in repression of the expression of class 1 KNOX and abaxial-determinant genes in the development of the adaxial domain of leaves and, thus, in the establishment of leaf polarity.