Expression of a mutant maize gene in the ventral leaf epidermis is sufficient to signal a switch of the leaf's dorsoventral axis

Maize leaves are initiated from the shoot apex with an inherent leaf dorsoventral polarity; the leaf surface closest to the meristem is the adaxial (upper, dorsal) surface whereas the opposite leaf surface is the abaxial (lower, ventral) surface. The Rolled leaf1 (Rld1) semi-dominant maize mutations affect dorsoventral patterning by causing adaxialization of abaxial leaf regions. This adaxialization is sometimes associated with abaxialization of the adaxial leaf regions, which constitutes a "switch". Dosage analysis indicates Rld1 mutants are antimorphs. We mapped Rld1's action to a single cell layer using a mosaic analysis and show Rld1 acts non cell-autonomously along the dorsoventral axis. The presence of Rld1 mutant product in the abaxial epidermis is necessary and sufficient to induce the Rolled leaf1 phenotype within the lower epidermis as well as in other leaf layers along the dorsoventral axis. These results support a model for the involvement of wild-type RLD1 in the maintenance of dorsoventral features of the leaf. In addition, they demonstrate the abaxial epidermis sends/receives a cell fate determining signal to/from the adaxial epidermis and controls the dorsoventral patterning of the maize leaf.     

Establishment of polarity in lateral organs of plants

BACKGROUND: Asymmetric development of plant lateral organs initiates by partitioning of organ primordia into distinct domains along their adaxial/abaxial axis. A recent model proposes that a meristem-born signal, acting in a concentration-dependent manner, differentially activates PHABULOSA-like genes, which in turn suppress abaxial-promoting factors. As yet, no abaxial factors have been identified that when compromised give rise to adaxialized organs. RESULTS: Single mutants in either of the closely related genes KANADI1 (KAN1) or KANADI2 (KAN2) have little or no effect on plant morphology. However, in kan1 kan2 double mutant plants, there is a replacement of abaxial cell types by adaxial ones in most lateral organs. The alterations in polarity establishment are associated with expansion in the expression domain of the PHB-like genes and reduction in the expression of the previously described abaxial-promoting YABBY genes. Ectopic expression of either of the KANADI genes throughout leaf primordia results in dramatic transformation of adaxial cell types into abaxial ones, failure of lateral blade expansion, and vascular tissue formation. CONCLUSION: The phenotypes of KANADI loss- and gain-of-function alleles suggest that fine regulation of these genes is at the core of polarity establishment. As such, they are likely to be targets of the PHB-mediated meristem-born signaling that patterns lateral organ primordia. PHB-like genes and the abaxial-promoting KANADI and YABBY genes appear to be expressed throughout primordia anlagen before becoming confined to their corresponding domains as primordia arise. This suggests that the establishment of polarity in plant lateral organs occurs via mutual repression interactions between ab/ad factors after primordium emergence, consistent with the results of classical dissection experiments.     

The maize milkweed pod1 mutant reveals a mechanism to modify organ morphology

Plant lateral organs, such as leaves, have three primary axes of growth–proximal‐distal, medial‐‐lateral and adaxial‐abaxial (dorsal‐ventral). Although most leaves are planar, modified leaf forms, such as the bikeeled grass prophyll, can be found in nature. A detailed examination of normal prophyll development indicates that polarity is established differently in the keels than in other parts of the prophyll. Analysis of the maize HD‐ZIPIII gene rolled leaf1 (rld1) suggests that altered expression patterns are responsible for keel outgrowth. Recessive mutations in the maize (Zea mays) KANADI (KAN) gene milkweed pod1 (mwp1), which promotes abaxial cell identity, strongly affect development of the prophyll and silks (fused carpels). The prophyll is reduced to two unfused midribs and the silks are narrow and misshapen. Our data indicate that the prophyll and other fused organs are particularly sensitive to disruptions in adaxial‐abaxial polarity. In addition, lateral and proximal‐distal growth of most lateral organs is reduced in the mwp1‐R mutant, supporting a role for the adaxial‐abaxial boundary in promoting growth along both axes. We propose that the adaxial‐abaxial patterning mechanism has been co‐opted during evolution to generate diverse organ morphologies. genesis 48:416–423, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

The formation and patterning of leaves: recent advances

Leaves, the plants major photosynthetic organs, form through the activity of groups of pluripotent cells, termed shoot apical meristems (SAMs), located at the growing tips of plants. Leaves develop with a dorso–ventral asymmetry, with the adaxial surface adjacent to the meristem and the abaxial surface developing at a distance from it. Molecular genetic studies have shown that the correct specification of adaxial/abaxial polarity requires communication between the incipient leaf and the meristem, and that the juxtaposition of adaxial/abaxial fates is necessary for lamina outgrowth (Waites and Hudson 1995; McConnell et al. 2001). Over the last few years, a number of factors that control cell fate specification in the apex have been identified. This review will focus on recent advances on distinct but overlapping aspects of leaf development, namely, the transition from meristem to leaf fate and the specification of abaxial/adaxial polarity and its possible role in leaf growth.     

Biomechanical analysis of the Rolled (RLD) leaf phenotype of maize

The pleiotropic effects of the Rld1-O/+ mutation of Zea mays (Poaceae) on leaf phenotype include a suppression of normal transverse unrolling, a reversed top/bottom epidermal polarity, and an apparently straighter longitudinal shape. According to engineering shell theory, there might be mechanical coupling between transverse and longitudinal habit, i.e., the leaf rolling itself might produce the longitudinal straightening. We tested this possibility with quantitative curvature measurements and mechanical uncoupling experiments. The contributions of elastic bending under self weight, mechanical coupling, and rest state of leaf parts to the longitudinal and transverse habit were assessed in Rld1-O/+ mutants and a population of sibling +/+ segregants. Elastic bending and curvature coupling are shown to be relatively unimportant. The Rld1-O/+ mutation is shown to alter not only the unrolling process, but also the developmental longitudinal curving in the growing leaf, leading to a straighter midrib and a rolled lamina. The Rld1-O/+ mutant is thus a suitable model to study the relation between tissue polarity and differential curvature development in the maize leaf. Since on the abaxial side of the leaf, more abundant sclerenchyma is found in +/+ than in Rld1-O/+, a gradient in sclerification may contribute to the development of midrib curvature.     

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.     

Ficus rubiginosa 'variegata', a chlorophyll-deficient chimera with mosaic patterns created by cell divisions from the outer meristematic layer

BACKGROUND AND AIMS: Sections leaves of Ficus rubiginosa 'Variegata' show that it is a chimera with a chlorophyll deficiency in the second layer of the leaf meristem (GWG structure). Like other Ficus species, it has a multiseriate epidermis on the adaxial and abaxial sides of the leaf, formed by periclinal cell divisions as well as anticlinal divisions. The upper and lower laminae of the leaf often exhibit small dark and light green patches of tissue overlying internal leaf tissue. METHODS: The distribution of chlorophyll in transverse sections of typical leaves was determined by fluorescence microscopy. KEY RESULTS: Patches of dark and light green tissue which arise in the otherwise colourless palisade and spongy mesophyll tissue in the entire leaf are due to further cell divisions arising from the bundle sheath which is associated with major vascular bundles or from the green multiseriate epidermis. Leaves produced in winter exhibit more patches of green tissue than leaves which expand in mid-summer. Many leaves produced in summer have no spotting and appear like a typical GWG chimera. There is a strong relationship between the number of patches on the adaxial side of leaves and the number on the abaxial side, showing that the cell division in upper and lower layers of leaves is strongly coordinated. In both winter and summer, there are fewer patches on the abaxial side of leaves compared with the adaxial side, indicating that periclinal and anticlinal cell divisions from the outer meristematic layer are less frequent in the lower layers of leaf tissue. Most of the patches are small (<1 mm in longest dimension) and thus the cell divisions which form them occur late in leaf development. Leaves which exhibit large patches generally have them on both sides of the leaves. CONCLUSION: In this cultivar, the outer meristematic layer appears to form vascular bundle sheaths and associated internal leaf tissue in the entire leaf lamina.     

Cell lineage analysis of maize bundle sheath and mesophyll cells

Maize leaves are divided into repeated longitudinal units consisting of vascular tissue, bundle sheath (BS), and mesophyll (M) cells. We have carried out a cell lineage analysis of these cell types using six spontaneous striping mutants of maize. We show that certain cell division patterns are preferentially utilized, but not required, to form the characteristic arrangement of cell types. Our data suggest that early in development a central cell layer is formed, most frequently by periclinal divisions in the adaxial subepidermal layer of the leaf primordium. Lateral and intermediate veins are initiated in this central layer, most often by divisions which contribute daughter cells to both the procambium and the ground meristem. These divisions generate "half vein" units which comprise half of the bundle sheath cells around a vein and a single adjacent M cell. We show that intermediate veins are multiclonal both in this transverse direction and along their lengths. BS cells are more closely related to M cells in the middle layer of the leaf than to those in the upper and lower subepidermal layers. An examination of sector boundaries has shown that photosynthetic differentiation in M cells is affected by the phenotype of neighboring BS cells.     

Transforming compound leaf patterning by manipulating REVOLUTA in Medicago truncatula

Leaves are derived from the shoot apical meristem with three distinct axes: dorsoventral, proximodistal and mediolateral. Different regulators are involved in the establishment of leaf polarity. Members of the class III homeodomain‐leucine zipper (HD‐ZIPIII) gene family are critical players in the determination of leaf adaxial identity mediated by microRNA165/166. However, their roles in compound leaf development are still unclear. By screening of a retrotransposon‐tagged mutant population of the model legume plant Medicago truncatula, a mutant line with altered leaflet numbers was isolated and characterized. Mutant leaves partially lost their adaxial identity. Leaflet numbers in the mutant were increased along the proximodistal axis, showing pinnate pentafoliate leaves in most cases, in contrast to the trifoliate leaves of the wild type. Detailed characterization revealed that a lesion in a HD‐ZIPIII gene, REVOLUTA (MtREV1), resulted in the defects of the mutant. Overexpression of MtMIR166‐insensitive MtREV1 led to adaxialized leaves and ectopic leaflets along the dorsoventral axis. Accompanying the abnormal leaf patterning, the free auxin content was affected. Our results demonstrate that MtREV1 plays a key role in determination of leaf adaxial–abaxial polarity and compound leaf patterning, which is associated with proper auxin homeostasis.     

Radial patterning of Arabidopsis shoots by class III HD-ZIP and KANADI genes

BACKGROUND: Shoots of all land plants have a radial pattern that can be considered to have an adaxial (central)-abaxial (peripheral) polarity. In Arabidopsis, gain-of-function alleles of PHAVOLUTA and PHABULOSA, members of the class III HD-ZIP gene family, result in adaxialization of lateral organs. Conversely, loss-of-function alleles of the KANADI genes cause an adaxialization of lateral organs. Thus, the class III HD-ZIP and KANADI genes comprise a genetic system that patterns abaxial-adaxial polarity in lateral organs produced from the apical meristem. RESULTS: We show that gain-of-function alleles of REVOLUTA, another member of the class III HD-ZIP gene family, are characterized by adaxialized lateral organs and alterations in the radial patterning of vascular bundles in the stem. The gain-of-function phenotype can be obtained by changing only the REVOLUTA mRNA sequence and without changing the protein sequence; this finding indicates that this phenotype is likely mediated through an interference with microRNA binding. Loss of KANADI activity results in similar alterations in vascular patterning as compared to REVOLUTA gain-of-function alleles. Simultaneous loss-of-function of PHABULOSA, PHAVOLUTA, and REVOLUTA abaxializes cotyledons, abolishes the formation of the primary apical meristem, and in severe cases, eliminates bilateral symmetry; these phenotypes implicate these three genes in radial patterning of both embryonic and postembryonic growth. CONCLUSIONS: Based on complementary vascular and leaf phenotypes of class III HD-ZIP and KANADI mutants, we propose that a common genetic program dependent upon miRNAs governs adaxial-abaxial patterning of leaves and radial patterning of stems in the angiosperm shoot. This finding implies that a common patterning mechanism is shared between apical and vascular meristems.     

HYL1 regulates the balance between adaxial and abaxial identity for leaf flattening via miRNA-mediated pathways

HYPONASTIC LEAVES1 (HYL1) is an important regulator of microRNA (miRNA) biogenesis. Incurvature of rosette leaves in loss-of-function mutants of HYL1 implicates the regulation of leaf flatness by HYL1 via miRNA pathways. Recent studies have identified jba-1D, jaw-1D, and oe-160c, the dominant mutants of MIR166g, MIR319a, and MIR160c genes, respectively, which display three types of leaf curvature. However, it remains unclear whether or how HYL1 controls leaf flatness through the pathways mediated by these miRNAs. To define which miRNAs and target genes are relevant to the hyl1 phenotype in terms of leaf incurvature, the effects of three mutated MIRNA genes and their targets on the direction and extent of leaf curvature in hyl1 mutants were examined. The genetic analysis shows that the hyl1 phenotype is strongly rescued by jba-1D, but not by jaw-1D or oe-160c, whereas the mutant phenotypes of jba-1D, jaw-1D, or oe-160c leaves are compromised by the hyl1 allele. Expression analysis indicates that reduced accumulation of miR166, rather than of miR319a or miR160, causes incurvature of hyl1 leaves, and that miR319a-targeted TCP3 positively regulates the adaxial identity gene PHABULOSA while miR160-targeted ARF16 negatively regulates the abaxial identity gene FILAMENTOUS FLOWER. In these cases, the direction and extent of leaf incurvature are associated with the expression ratio of adaxial to abaxial genes (adaxial to abaxial ratio). HYL1 regulates the balance between adaxial and abaxial identity and modulates leaf flatness by preventing leaf incurvature, wavy margins, and downward curvature. It is concluded that HYL1 monitors the roles of miR165/166, miR319a, and miR160 in leaf flattening through the relative activities of adaxial and abaxial identity genes, thus playing an essential role in leaf development.     

BLADE-ON-PETIOLE 1 and 2 control Arabidopsis lateral organ fate through regulation of LOB domain and adaxial-abaxial polarity genes

We report a novel function for BLADE-ON-PETIOLE1 (BOP1) and BOP2 in regulating Arabidopsis thaliana lateral organ cell fate and polarity, through the analysis of loss-of-function mutants and transgenic plants that ectopically express BOP1 or BOP2. 35S:BOP1 and 35S:BOP2 plants exhibit a very short and compact stature, hyponastic leaves, and downward-orienting siliques. We show that the LATERAL ORGAN BOUNDARIES (LOB) domain genes ASYMMETRIC LEAVES2 (AS2) and LOB are upregulated in 35S:BOP and downregulated in bop mutant plants. Ectopic expression of BOP1 or BOP2 also results in repression of class I knox gene expression. We further demonstrate a role for BOP1 and BOP2 in establishing the adaxial-abaxial polarity axis in the leaf petiole, where they regulate PHB and FIL expression and overlap in function with AS1 and AS2. Interestingly, during this study, we found that KANADI1 (KAN1) and KAN2 act to promote adaxial organ identity in addition to their well-known role in promoting abaxial organ identity. Our data indicate that BOP1 and BOP2 act in cells adjacent to the lateral organ boundary to repress genes that confer meristem cell fate and induce genes that promote lateral organ fate and polarity, thereby restricting the developmental potential of the organ-forming cells and facilitating their differentiation.     

Nucleostemin-like 1 is required for embryogenesis and leaf development in Arabidopsis

Arabidopsis NSN1 encodes a nucleolar GTP-binding protein and is required for flower development. Defective flowers were formed in heterozygous nsn1/+?plants. Homozygous nsn1 plants were dwarf and exhibited severe defects in reproduction. Arrests in embryo development in nsn1 could occur at any stage of embryogenesis. Cotyledon initiation and development during embryogenesis were distorted in nsn1 plants. At the seedling stage, cotyledons and leaves of nsn1 formed upward curls. The curled leaves developed meristem-like outgrowths or hyperplasia tissues in the adaxial epidermis. Long and enlarged pavement cells, characteristic of the abaxial epidermis of wild type plants, were found in the adaxial epidermis in nsn1 leaves, suggesting a disoriented leaf polarity in the mutant. The important role of NSN1 in embryo development and leaf differentiation was consistent with the high level expression of the NSN1 gene in the developing embryos and the primordia of cotyledons and leaves. The CLAVATA 3 (CLV3) gene, a stem cell marker in the Arabidopsis shoot apical meristem (SAM), was expressed in expanded regions surrounding the SAM of nsn1 plants, and induced ectopically in the meristem-like outgrowths in cotyledons and leaves. The nsn1 mutation up-regulated the expression levels of several genes implicated in the meristem identity and the abaxial cell fate, and repressed the expression of other genes related to the specification of cotyledon boundary and abaxial identity. These results demonstrate that NSN1 represents a novel GTPase required for embryogenesis, leaf development and leaf polarity establishment in Arabidopsis.     

Developmental study onHypolepis punctata (Thunb.) Mett.

The origins of the first and second petiolar buds ofHypolepis punctata were clarified in relation to the early development of the leaf primordium, which arises from a group of superficial cells of the shoot apical meristem. One of these superficial cells produces a two-sided leaf apical cell which subsequently cuts off segments to make a well-defined cell group, called here the leaf apical cell complex, on the distal part of the leaf primordium. Meanwhile, cells surrounding the leaf apical cell complex also divide frequently to form the basal part of the leaf primordium. Two groups of basal cells of the leaf primordium located on the abaxial and the adaxial sides initiate the first and the second petiolar buds, respectively. The initial cells are usually contiguous to the leaf apical cell complex, constructing the abaxial and adaxial flanks of the very young leaf primordium. However, the first petiolar bud sometimes develops from cells located farther from the leaf apical cell complex. These cells are derived from those originally situated in the peripheral region of the shoot apical meristem. This study was supported by a Grant-in-Aid for Encouragement of Young Scientists by the Ministry of Education, Science and Culture, of Japan No. 474322 in 1979.