Research Summary Molecular Genetics of Plant Morphogenesis The vast majority of plant developmental patterning and morphogenesis occurs postembryonically in meristems. Cells toward the center of the shoot apical meristem (SAM) form undifferentiated stem cells that divide and contribute both to maintaining the stem cell pool and to organ formation. Determinate lateral organs such as leaves and floral organs are initiated around the periphery of the SAM. Leaves and other lateral organs, therefore, have an inherent positional relationship with the SAM. This relationship defines the adaxial-abaxial axis of the leaf, otherwise known as the dorsal-ventral axis. Organ primordia form with their adaxial (or dorsal) side toward the meristem and their abaxial (or ventral) side away from the meristem axis. Recent genetic studies indicate that the proper establishment of adaxial-abaxial polarity within organ primordia is critical for the polarized growth and differentiation of the primordium and contributes to both the external morphology and internal anatomy of the mature leaf. In Arabidopsis, as in most plants, there are marked differences in the morphology of the adaxial and abaxial sides of leaves. In fully expanded leaves, the adaxial side of the leaf appears dark green, glossy, and bears many epidermal hairs or trichomes. In contrast, the abaxial side is gray-green, matte, and has fewer trichomes especially on juvenile leaves. The polarization of the leaf blade along its adaxial-abaxial axis is particularly evident in its internal anatomy. Closely packed palisade mesophyll forms just below the adaxial epidermis and loosely packed spongy mesophyll with large intercellular air spaces forms next to the abaxial epidermis. These asymmetries reflect functional specialization of the leaf surfaces: the adaxial (or top) for light harvesting and photosynthesis, and the abaxial (or bottom) for gas exchange. As the vascular system develops in the center of the leaf, xylem, which transports water and minerals, is formed adaxial to phloem, which transports sugars and other products of photosynthesis. Some polar traits appear to be determined very early in leaf development, whereas others develop quite late indicating that polarity, once established, must be maintained or propagated throughout leaf development. We are taking a molecular genetic approach to understand the mechanisms that regulate the specification of adaxial-abaxial polarity during leaf development. The KANADI 1 (KAN1) gene is a key promoter of abaxial leaf fate. KAN1 expression is first detected in the periphery of the globular embryo just as the basic plant body plan is being established. In the SAM, KAN1 expression is detected on the abaxial side of young leaf and floral organ primordia. Loss of function mutations in KAN1 result in a partial loss of abaxial identity during leaf development. Specific traits that are normally restricted to the adaxial side of the leaf, such as small intercellular air spaces (characteristic of palisade mesophyll) and epidermal hairs are expressed on both sides of mutant leaves. Other polarity-related traits remain unaffected in kan1 mutants—leaf shape remains normal and the epidermal cells retain their normal ad- and abaxial distinctiveness. When KAN1 is misexpressed under the control of a strong constitutive promoter, elongated, nearly radially symmetric embryonic leaves are formed and fundamental patterning of the shoot is disrupted: the central vascular strand of the embryonic stem fails to form, and the shoot apical meristem is absent. This phenotype indicates a profound abaxialization of the shoot and a loss of fates associated with the central meristematic domain of the embryo. KAN1 encodes a putative transcription factor of the GARP domain family. The GARP domain is a plant-specific, conserved DNA binding domain of the helix-turn-helix superfamily that is distantly related to the MYB domain. GARP is an acronym for the founding members of the family: maize Golden 2, ARR (Arabidopsis Response Regulators) and Psr1 (phosphorous stress response1 from Chlamydomonas). Suppressors and Enhancers of the kanadi1 Mutant Phenotype To identify new genes that affect the specification of adaxial or abaxial fate in leaves, we have been screening for enhancers and suppressors the kan1 mutant phenotype. We have identified a dominant kan1 suppressor, Doks1. Plants homozygous for kan loss of function alleles and heterozygous for Doks1 are indistinguishable from wild-type plants. Plants homozygous for both kan and Doks1 display a dramatic and novel leaf shape phenotype. The novel recessive mutant is characterized by small, elongated, and sometimes, asymmetrical leaves and a lighter green color (similar to the appearance of abaxial leaf tissue). We are pursuing a map based approach to clone the Doks1 gene. We have narrowed the region to a 0.5 Megabase region on chromosome V and are currently generating new recombinants using marked chromosomes in order to get closer to the gene responsible for the Doks1 mutant. One of the enhancers of kan that has caught our interest causes outgrowths of leaf-like tissue from the abaxial leaf surface. Since the botanical term for a leaf outgrowth is “prickle”, we have dubbed the mutant prickly. Plants doubly mutant for kan1 and prickly show dramatically altered floral structures such as radialized petals and stamens and naked ovules where the pistil is normally formed. The prickly mutant phenotype shows some similarity to a weak allele of argonaute 1, a gene involved in the processing of microRNAs. We are currently testing for allelism with argonaute, characterizing the polarity defects in the double mutant, and generating a mapping population in order to clone prickly should it turn out not to be an allele of argonaute. The ongoing suppressor and enhancer screens have provided a rich source of potential new leaf polarity mutants that are at a very preliminary stage in their characterization. Mutants with radialized leaves, disturbed margins, and a variety of other defects in leaf morphogenesis have been identified with the aim of mapping and, eventually, cloning the genes underlying the defect in leaf development. GAL4-UAS Enhancer Trap Lines for Activation Tagging Mutagenesis In order to identify and manipulate additional genetic determinants of leaf polarity, we are taking advantage of a set of transgenic tools for targeted gene expression, based on a method widely used in Drosophila. An "enhancer-trap" strategy was employed to generate many transgenic plants that express a yeast transcription activator, GAL4 in different tissue- or cell-specific patterns. A gene-of-interest (GOI) can then be placed under the control of GAL4 upstream activation sequences (UAS), transformed into plants, and maintained silently in the absence of GAL4. Genetic crosses between this line and any of the GAL4-containing lines specifically activates the target gene in a particular tissue or cell type. The “enhancer trap” vector was designed so that expression of the GAL4 transgene depends upon proximity to an Arabidopsis enhancer element. GAL4 expression is detected with a linked GAL4-responsive green fluorescent protein (GFP) gene, so that each GAL4-expressing cell is marked by green fluorescence. The enhancer trap lines that express GAL4 are used for targeted gene expression experiments. We have produced transgenic plants that maintain marker genes, regulatory proteins, or toxins silent behind a GAL4-responsive promoter. These genes are activated in specific cells by crossing to a GAL4 enhancer trap line. The phenotypic consequences of the GAL4-regulated GOI expression can be conveniently studied. Several enhancer trap lines display differential GFP expression in either adaxial or abaxial tissues. These lines provide a valuable set of markers for examining the effects of mutants that disrupt normal leaf polarity. In addition, they provide a jumping off point for identifying new genes involved in leaf morphogenesis. I have produced a set of T-DNA vectors that allow the introduction of an outwardly directed GAL4-responsive UAS enhancer. This vector allows us to subvert the normal transcriptional regulation of any gene near the site of the T-DNA insertion by bringing it under the influence of GAL4. When GAL4 is active, the tagged gene is activated. With different GAL4 enhancer trap lines, we should be able to drive expression of the tagged gene in a wide variety of tissues and cell types. We are currently screening for morphological mutants in a population of UAS “activation tag” insertion mutants in which GAL4 is expressed in abaxial tissues. We expect to identify novel genes whose misexpression in abaxial tissues perturbs normal leaf polarity. A subset of such genes may play significant roles in normal pattern formation during development. Although encouraged by initially finding many novel morphological mutants in the pilot UAS activation tagging population, most fail to show the parental phenotype among their progeny. This result may indicate either that most of the mutants do not carry heritable mutations or that the ectopic expression driven by the UAS transgene is being silenced. We are currently testing the role of siRNA in the silencing of UAS activation tagged mutants and are redesigning the mutagenesis strategy to minimize the effects of transgene silencing. In the meantime, we are re-screening the selfed progeny of the original mutants for those with interesting and heritable phenotypes that may lead us to new players in the establishment or maintenance of leaf polarity. Publications Lab Support Yael Harrar, Postdoctoral Researcher Jeon J. Hong, Laboratory Assistant Sean Chen, Undergraduate Researcher