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.     

The proteolytic function of the Arabidopsis 26S proteasome is required for specifying leaf adaxial identity

Polarity formation is central to leaf morphogenesis, and several key genes that function in adaxial-abaxial polarity establishment have been identified and characterized extensively. We previously reported that Arabidopsis thaliana ASYMMERTIC LEAVES1 (AS1) and AS2 are important in promoting leaf adaxial fates. We obtained an as2 enhancer mutant, asymmetric leaves enhancer3 (ae3), which demonstrated pleiotropic plant phenotypes, including a defective adaxial identity in some leaves. The ae3 as2 double mutant displayed severely abaxialized leaves, which were accompanied by elevated levels of leaf abaxial promoting genes FILAMENTOUS FLOWER, YABBY3, KANADI1 (KAN1), and KAN2 and a reduced level of the adaxial promoting gene REVOLUTA. We identified AE3, which encodes a putative 26S proteasome subunit RPN8a. Furthermore, double mutant combinations of as2 with other 26S subunit mutations, including rpt2a, rpt4a, rpt5a, rpn1a, rpn9a, pad1, and pbe1, all displayed comparable phenotypes with those of ae3 as2, albeit with varying phenotypic severity. Since these mutated genes encode subunits that are located in different parts of the 26S proteasome, it is possible that the proteolytic function of the 26S holoenzyme is involved in leaf polarity formation. Together, our findings reveal that posttranslational regulation is essential in proper leaf patterning.     

Specification of leaf polarity in Arabidopsis via the trans-acting siRNA pathway

Plants leaves develop proximodistal, dorsoventral (adaxial-abaxial), and mediolateral patterns following initiation. The Myb domain gene PHANTASTICA (PHAN) is required for adaxial fate in many plants , but the Arabidopsis ortholog ASYMMETRIC LEAVES1 (AS1) has milder effects, suggesting that alternate or redundant pathways exist . We describe enhancers of as1 with more elongate and dissected leaves. As well as RDR6, an RNA-dependent RNA polymerase previously proposed to influence as1 through microRNA , these enhancers disrupt ARGONAUTE7 (AGO7)/ZIPPY, SUPPRESSOR OF GENE SILENCING3 (SGS3), and DICER-LIKE4 (DCL4), which instead regulate trans-acting small interfering RNA (ta-siRNA) . Microarray analysis revealed that the AUXIN RESPONSE FACTOR genes ETTIN (ETT)/ARF3 and ARF4 were upregulated in ago7, whereas FILAMENTOUS FLOWER (FIL) was upregulated only in as1 ago7 double mutants. RDR6 and SGS3 likewise repress these genes, which specify abaxial fate . We show that the trans-acting siRNA gene TAS3, which targets ETT and ARF4, is expressed in the adaxial domain, and ett as1 ago7 triple mutants resemble as1. Thus FIL is downregulated redundantly by AS1 and by TAS3, acting through ETT, revealing a role for ta-siRNA in leaf polarity. RDR6 and DCL4 are required for systemic silencing, perhaps implicating ta-siRNA as a mobile signal.     

Novel as1 and as2 defects in leaf adaxial-abaxial polarity reveal the requirement for ASYMMETRIC LEAVES1 and 2 and ERECTA functions in specifying leaf adaxial identity

The shoot apical meristem (SAM) of seed plants is the site at which lateral organs are formed. Once organ primordia initiate from the SAM, they establish polarity along the adaxial-abaxial, proximodistal and mediolateral axes. Among these three axes, the adaxial-abaxial polarity is of primary importance in leaf patterning. In leaf development, once the adaxial-abaxial axis is established within leaf primordia, it provides cues for proper lamina growth and asymmetric development. It was reported previously that the Arabidopsis ASYMMETRIC LEAVES1 (AS1) and ASYMMETRIC LEAVES2 (AS2) genes are two key regulators of leaf polarity. In this work, we demonstrate a new function of the AS1 and AS2 genes in the establishment of adaxial-abaxial polarity by analyzing as1 and as2 alleles in the Landsberg erecta (Ler) genetic background. We provide genetic evidence that the Arabidopsis ERECTA (ER) gene is involved in the AS1-AS2 pathway to promote leaf adaxial fate. In addition, we show that AS1 and AS2 bind to each other, suggesting that AS1 and AS2 may form a complex that regulates the establishment of leaf polarity. We also report the effects on leaf polarity of overexpression of the AS1 or AS2 genes under the control of the cauliflower mosaic virus (CAMV) 35S promoter. Although plants with as1 and as2 mutations have very similar phenotypes, 35S::AS1/Ler and 35S::AS2/Ler transgenic plants showed dramatically different morphologies. A possible model of the AS1, AS2 and ER action in leaf polarity formation is discussed.     

Adaxial–abaxial polarity: The developmental basis of leaf shape diversity

Leaves of flowering plants are diverse in shape. Part of this morphological diversity can be attributed to differences in spatiotemporal regulation of polarity in the upper (adaxial) and lower (abaxial) sides of developing leaves. In a leaf primordium, antagonistic interactions between polarity determinants specify the adaxial and abaxial domains in a mutually exclusive manner. The patterning of those domains is critical for leaf morphogenesis. In this review, we first summarize the gene networks regulating adaxial–abaxial polarity in conventional bifacial leaves and then discuss how patterning is modified in different leaf type categories. genesis 52:1–18, 2014. © 2013 The Authors. Genesis Published byWiley Periodicals, Inc.     

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.     

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.     

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

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.