The maize preligule band is subdivided into distinct domains with contrasting cellular properties prior to ligule outgrowth.

The maize ligule is an epidermis-derived structure that arises from the preligule band (PLB) at a boundary between the blade and sheath. A hinge-like auricle also develops immediately distal to the ligule and contributes to blade angle. Here, we characterize the stages of PLB and early ligule development in terms of topography, cell area, division orientation, cell wall rigidity and auxin response dynamics. Differential thickening of epidermal cells and localized periclinal divisions contributed to the formation of a ridge within the PLB, which ultimately produces the ligule fringe. Patterns in cell wall rigidity were consistent with the subdivision of the PLB into two regions along a distinct line positioned at the nascent ridge. The proximal region produces the ligule, while the distal region contributes to one epidermal face of the auricles. Although the auxin transporter PIN1 accumulated in the PLB, observed differential auxin transcriptional response did not underlie the partitioning of the PLB. Our data demonstrate that two zones with contrasting cellular properties, the preligule and preauricle, are specified within the ligular region before ligule outgrowth.

Arabidopsis lateral organ boundaries negatively regulates brassinosteroid accumulation to limit growth in organ boundaries.

Leaves and flowers begin life as outgrowths from the edges of shoot apical meristems. Stem cell divisions in the meristem center replenish cells that are incorporated into organ primordia at the meristem periphery and leave the meristem. Organ boundaries, regions of limited growth that separate forming organs from the meristem, serve to isolate these two domains and are critical for coordination of organogenesis and meristem maintenance. Boundary formation and maintenance are poorly understood processes, despite the identification of a number of boundary-specific transcription factors. Here we provide genetic and biochemical evidence that the Arabidopsis thaliana transcription factor lateral organ boundaries (LOB) negatively regulates accumulation of the plant steroid hormone brassinosteroid (BR) in organ boundaries. We found that ectopic expression of LOB results in reduced BR responses. We identified BAS1, which encodes a BR-inactivating enzyme, as a direct target of LOB transcriptional activation. Loss-of-function lob mutants exhibit organ fusions, and this phenotype is suppressed by expression of BAS1 under the LOB promoter, indicating that BR hyperaccumulation contributes to the lob mutant phenotype. In addition, LOB expression is BR regulated; therefore, LOB and BR form a feedback loop to modulate local BR accumulation in organ boundaries to limit growth in the boundary domain.

Brassinosteroids regulate organ boundary formation in the shoot apical meristem of Arabidopsis.

Spatiotemporal control of the formation of organ primordia and organ boundaries from the stem cell niche in the shoot apical meristem (SAM) determines the patterning and architecture of plants, but the underlying signaling mechanisms remain poorly understood. Here we show that brassinosteroids (BRs) play a key role in organ boundary formation by repressing organ boundary identity genes. BR-hypersensitive mutants display organ-fusion phenotypes, whereas BR-insensitive mutants show enhanced organ boundaries. The BR-activated transcription factor BZR1 directly represses the cup-shaped cotyledon (CUC) family of organ boundary identity genes. In WT plants, BZR1 accumulates at high levels in the nuclei of central meristem and organ primordia but at a low level in organ boundary cells to allow CUC gene expression. Activation of BR signaling represses CUC gene expression and causes organ fusion phenotypes. This study uncovers a role for BR in the spatiotemporal control of organ boundary formation and morphogenesis in the SAM.

Misregulation of the LOB domain gene DDA1 suggests possible functions in auxin signalling and photomorphogenesis.

The LATERAL ORGAN BOUNDARIES DOMAIN (LBD) gene family encodes plant-specific transcription factors. In this report, the LBD gene DOWN IN DARK AND AUXIN1 (DDA1), which is closely related to LATERAL ORGAN BOUNDARIES (LOB) and ASYMMETRIC LEAVES2 (AS2), was characterized. DDA1 is expressed primarily in vascular tissues and its transcript levels were reduced by exposure to exogenous indole-3-acetic acid (IAA or auxin) and in response to dark exposure. Analysis of a T-DNA insertion line, dda1-1, in which the insertion resulted in misregulation of DDA1 transcripts in the presence of IAA and in the dark revealed possible functions in auxin response and photomorphogenesis. dda1-1 plants exhibited reduced sensitivity to auxin, produced fewer lateral roots, and displayed aberrant hypocotyl elongation in the dark. Phenotypes resulting from fusion of a transcriptional repression domain to DDA1 suggest that DDA1 may act as both a transcriptional activator and a transcriptional repressor depending on the context. These results indicate that DDA1 may function in both the auxin signalling and photomorphogenesis pathways.

Architecture of floral branch systems in maize and related grasses.

The external appearance of flowering plants is determined to a large extent by the forms of flower-bearing branch systems, known as inflorescences, and their position in the overall structure of the plant. Branches and branching patterns are produced by tissues called shoot apical meristems. Thus, inflorescence architecture reflects meristem number, arrangement and activity, and the duration of meristem activity correlates with branch length. The inflorescences of maize, unlike those of related grasses such as rice and sorghum, predominantly lack long branches, giving rise to the tassel and familiar corncob. Here we report the isolation of the maize ramosa1 gene and show that it controls inflorescence architecture. Through its expression in a boundary domain near the nascent meristem base, ramosa1 imposes short branch identity as branch meristems are initiated. A second gene, ramosa2, acts through ramosa1 by regulating ramosa1 gene expression levels. ramosa1 encodes a transcription factor that appears to be absent in rice, is heterochronically expressed in sorghum, and may have played an important role in maize domestication and grass evolution.

KANADI1 regulates adaxial-abaxial polarity in Arabidopsis by directly repressing the transcription of ASYMMETRIC LEAVES2.

Lateral organ polarity in Arabidopsis is regulated by antagonistic interactions between genes that promote either adaxial or abaxial identity, but the molecular basis of this interaction is largely unknown. We show that the adaxial regulator ASYMMETRIC LEAVES2 (AS2) is a direct target of the abaxial regulator KANADI1 (KAN1), and that KAN1 represses the transcription of AS2 in abaxial cells. Mutation of a single nucleotide in a KAN1 binding site in the AS2 promoter causes AS2 to be ectopically expressed in abaxial cells, resulting in a dominant, adaxialized phenotype. We also show that the abaxial expression of KAN1 is mediated directly or indirectly by AS2. These results demonstrate that KAN1 acts as a transcriptional repressor and that mutually repressive interactions between KAN1 and AS2 contribute to the establishment of adaxial-abaxial polarity in plants.

LATERAL ORGAN BOUNDARIES defines a new family of DNA-binding transcription factors and can interact with specific bHLH proteins.

Conserved in a variety of evolutionarily divergent plant species, LOB DOMAIN (LBD) genes define a large, plant-specific family of largely unknown function. LBD genes have been implicated in a variety of developmental processes in plants, although to date, relatively few members have been assigned functions. LBD proteins have previously been predicted to be transcription factors, however supporting evidence has only been circumstantial. To address the biochemical function of LBD proteins, we identified a 6-bp consensus motif recognized by a wide cross-section of LBD proteins, and showed that LATERAL ORGAN BOUNDARIES (LOB), the founding member of the family, is a transcriptional activator in yeast. Thus, the LBD genes encode a novel class of DNA-binding transcription factors. Post-translational regulation of transcription factors is often crucial for control of gene expression. In our study, we demonstrate that members of the basic helix-loop-helix (bHLH) family of transcription factors are capable of interacting with LOB. The expression patterns of bHLH048 and LOB overlap at lateral organ boundaries. Interestingly, the interaction of bHLH048 with LOB results in reduced affinity of LOB for the consensus DNA motif. Thus, our studies suggest that bHLH048 post-translationally regulates the function of LOB at lateral organ boundaries.

Ustilago maydis Infection of the Nonnatural Host Arabidopsis thaliana.

ABSTRACT The experimental infection of Arabidopsis thaliana by the maize phytopathogenic hemibasidiomycete Ustilago maydis under axenic conditions is described. When plantlets were inoculated with mixtures of compatible haploids, the fungus was able to grow on the plant surface of inoculated seedlings in the form of white mycelium and invade the tissues, probably penetrating through stomata; however, it did not form teliospores. Symptoms of disease were increased anthocyanin formation, development of chlorosis, increased formation of secondary roots, induction of malformations in the leaves and petioles, induction of tissue necrosis, and stunting. In several cases, death of the invaded plants occurred. Interestingly, inoculation of single U. maydis haploid strains produced similar symptoms in Arabidopsis plantlets. In contrast, several mutants avirulent to maize also were avirulent or less virulent than wildtype strains on Arabidopsis. Collectively, the reported data suggest that the U. maydis-Arabidopsis pathosystem may constitute a useful experimental model for the analysis of some aspects of the virulence factors of the fungus. With the study of nonhost responses and their comparison to those occurring during maize infection, we will be able to elucidate some obscure aspects of U. maydis pathogenicity in the future.

Gene and enhancer traps for gene discovery.

Gene traps and enhancer traps provide a valuable tool for gene discovery. With this system, genes can be identified based solely on the expression pattern of an inserted reporter gene. The use of a reporter gene, such as beta-glucuoronidase (GUS), provides a very sensitive assay for the identification of tissue- and cell-type specific expression patterns. In this chapter, protocols for examining and documenting GUS reporter gene activity in individual lines are described. Methods for the amplification of sequences flanking transposant insertions and subsequent molecular and genetic characterization of individual insertions are provided.

PLASTID MOVEMENT IMPAIRED1 mediates ABA sensitivity during germination and implicates ABA in light-mediated Chloroplast movements.

The plant hormone abscisic acid (ABA) controls many aspects of plant growth and development, including seed development, germination and responses to water-deficit stress. A complex ABA signaling network integrates environmental signals including water availability and light intensity and quality to fine-tune the response to a changing environment. To further define the regulatory pathways that control water-deficit and ABA responses, we carried out a gene-trap tagging screen for water-deficit-regulated genes in Arabidopsis thaliana. This screen identified PLASTID MOVEMENT IMPAIRED1 (PMI1), a gene involved in blue-light-induced chloroplast movement, as functioning in ABA-response pathways. We provide evidence that PMI1 is involved in the regulation of seed germination by ABA, acting upstream of the intersection between ABA and low-glucose signaling pathways. Furthermore, PMI1 participates in the regulation of ABA accumulation during periods of water deficit at the seedling stage. The combined phenotypes of pmi1 mutants in chloroplast movement and ABA responses indicate that ABA signaling may modulate chloroplast motility. This result was further supported by the detection of altered chloroplast movements in the ABA mutants aba1-6, aba2-1 and abi1-1.

Functional divergence in the Arabidopsis LOB-domain gene family.

The Arabidopsis LOB-domain (LBD) gene family is composed by 43 members divided in two classes based on amino acid conservation within the LOB-domain. The LOB domain is known to be responsible for DNA binding and protein-protein interactions. There is very little functional information available for most genes in the LBD family and many lbd single mutants do not exhibit conspicuous phenotypes. One plausible explanation for the limited loss-of-function phenotypes observed in this family is that LBD genes exhibit significant functional redundancy. Here we discuss an example of one phylogenetic subgroup of the LBD family, in which genes that are closely related based on phylogeny exhibit distinctly different expression patterns and do not have overlapping functions. We discuss the challenges of using phylogenetic analyses to predict redundancy in gene families.

LATERAL ORGAN FUSION1 and LATERAL ORGAN FUSION2 function in lateral organ separation and axillary meristem formation in Arabidopsis.

Plant organs are generated from meristems throughout development. Patterning and elaboration of organ primordia occur as a result of organized cell division and expansion, processes that are likely to be controlled, in part, by meristem-derived signals. Communication between the meristem and lateral organs is crucial for meristem maintenance and organ patterning, and organ boundaries are thought to be important for mediating this communication. Arabidopsis thaliana LATERAL ORGAN FUSION1 (LOF1) encodes a MYB-domain transcription factor that is expressed in organ boundaries. lof1 mutants display defects in organ separation as a result of abnormal cell division and expansion during early boundary formation. lof1 mutants also fail to form accessory shoot meristems. Mutations in the closely related LATERAL ORGAN FUSION2 (LOF2) gene enhance the lof1 phenotype, such that lof1 lof2 double mutants display additional fusion defects. Genetic interactions with the CUP-SHAPED COTYLEDON genes CUC2 and CUC3 revealed a role for LOF1 in both organ separation and axillary meristem formation. Expression of the meristem determinant STM was reduced in lof1 mutant paraclade junctions and lof1 enhanced the weak stm-10 mutant, such that double mutants had severe defects in meristem maintenance and organ separation. Our data implicate LOF1 and LOF2 in boundary specification, meristem initiation and maintenance, and organ patterning.

The Arabidopsis gene PROLIFERA is required for proper cytokinesis during seed development.

The Arabidopsis thaliana (L.) Heynh. gene PROLIFERA (PRL) is a member of the MCM family of genes that are required for DNA replication during the S phase of the cell cycle. PRL is expressed in dividing cells throughout plant development. During reproductive development, PRL is expressed in both the developing megaspore mother cells and microspore mother cells, but is not expressed in the developing microgametophyte, suggesting that it does not function in the final haploid divisions leading to the production of a mature pollen grain. Disruption of PRL leads to megagametophyte and embryo lethality. prl mutant embryos arrest at a variety of stages, and often show defects in cytokinesis. Multinucleate cells and non-stereotypical cell division planes are commonly observed in developing prl mutant embryos, although mcm mutations in other organisms have not been reported to affect cytokinesis. These observations suggest that PRL may play a role in cytokinesis that is distinct from its role in regulating DNA replication. Additionally, a novel cytokinesis checkpoint that monitors cell cycle progression may exist in Arabidopsis.

The vascular prepattern enhancer trap marks early vascular development in arabidopsis.

Vascular development is a fundamental component of leaf morphogenesis, and the mechanisms that control vascular patterning are poorly understood. We report here the identification of an enhancer trap line, Vascular Prepattern (VPP), that acts as a marker for early vascular development. GUS reporter gene expression in VPP was detected in provascular cells from the earliest stages of primary midvein formation in leaf primordia and subsequently coincided with the early specification of higher order veins. GUS expression in VPP also marks the quiescent center cells of the root apical meristem at all stages of root development. VPP provides a marker for early vascular development and will be a useful tool for studying vascular patterning.

The Arabidopsis LATERAL ORGAN BOUNDARIES-domain gene ASYMMETRIC LEAVES2 functions in the repression of KNOX gene expression and in adaxial-abaxial patterning.

The normal development of lateral organs of the shoot requires the simultaneous repression of meristem-specific genes and the activation of organ-specific genes. ASYMMETRIC LEAVES2 (AS2) is required for the development of normal leaf shape and for the repression of KNOX genes in the leaf. AS2 is a member of the recently identified, plant-specific LATERAL ORGAN BOUNDARIES (LOB)-domain gene family. Expression of AS2 at high levels resulted in repression of the KNOX homeobox genes BREVIPEDICELLUS, KNAT2, and KNAT6 but not of the related SHOOT MERISTEMLESS gene. Overexpression of AS2 also led to a perturbation of normal adaxial-abaxial asymmetry in lateral organs, resulting in the replacement of abaxial cell types with adaxial cell types. These results indicate that AS2 is sufficient to induce adaxial cell fate and repress KNOX gene expression.

The lateral organ boundaries gene defines a novel, plant-specific gene family.

The LATERAL ORGAN BOUNDARIES (LOB) gene in Arabidopsis defines a new conserved protein domain. LOB is expressed in a band of cells at the adaxial base of all lateral organs formed from the shoot apical meristem and at the base of lateral roots. LOB encodes a predicted protein that does not have recognizable functional motifs, but that contains a conserved domain (the LOB domain) that is present in 42 other Arabidopsis proteins and in proteins from a variety of other plant species. Proteins showing similarity to the LOB domain were not found outside of plant databases, indicating that this unique protein may play a role in plant-specific processes. Genes encoding LOB domain proteins are expressed in a variety of temporal- and tissue-specific patterns, suggesting that they may function in diverse processes. Loss-of-function LOB mutants have no detectable phenotype under standard growth conditions, suggesting that LOB is functionally redundant or required during growth under specific environmental conditions. Ectopic expression of LOB leads to alterations in the size and shape of leaves and floral organs and causes male and female sterility. The expression of LOB at the base of lateral organs suggests a potential role for LOB in lateral organ development.

Enhancer trap expression patterns provide a novel teaching resource.

A collection of Arabidopsis enhancer trap transposants has been identified for use as a teaching tool. This collection serves to assist students in understanding the patterning and organization of plant tissues and cells, and will be useful in plant anatomy, morphology, and developmental biology courses. Each transposant exhibits reporter gene expression in a specific tissue, cell type, or domain, and these lines collectively offer a glimpse of compartments of gene expression. Some compartments correspond to classical definitions of botanical anatomy and can assist in anatomical identification. Other patterns of reporter gene expression are more complex and do not necessarily correspond to known anatomical features. The sensitivity of the beta-glucuronidase histochemical stain provides the student with a colorful and direct way to visualize difficult aspects of plant development and anatomy, and provides the teacher with an invaluable tool for a practical laboratory session.

Structure and evolution of the Cinful retrotransposon family of maize.

A maize cDNA clone was isolated by virtue of its intense hybridization to total maize genomic DNA, indicating homology to highly repetitive sequences. Genomic homologues were identified and subcloned from an adh1-bearing maize yeast artificial chromosome (YAC). Sequencing revealed that the expressed sequence was part of a Ty3-gypsy-type retrotransposon. We discovered and sequenced two complete retrotransposons of this family, and named them Cinful elements because they are members of a family of maize retrotransposons including Zeon-1 and the first plant transposable element sequenced, the solo long terminal repeat (LTR) called Cin1. All are defective, as Cinful-1 and Cinful-2 elements lack gag and Zeon-1 lacks pol homology. Despite the apparent lack of an intact "autonomous" element, the Cinful family has expanded to a copy number of about 18 000, representing just under 9% of the maize genome. Both point mutations and major rearrangements, including possible gene acquisition, differentiate members of the Cinful family. Cinful family members were found to have an unusual feature that we also observed in two other Ty3-class retrotransposons of teosinte and tobacco: related tandem repeats that separate their internal domains with a gag- or pol-containing homology from a 3' segment of unknown function. The conserved and variable features identified provide insights into the origin, mutational history, and functional components of this major constituent of the maize genome.

A resource of mapped dissociation launch pads for targeted insertional mutagenesis in the Arabidopsis genome.

We describe a new resource for targeted insertional mutagenesis in Arabidopsis using a maize (Zea mays) Activator/Dissociation (Ds) two-element system. The two components of the system, T-DNA vectors carrying a Ds launch pad and a stable Activator transposase source, were designed to simplify selection of transposition events and maximize their usefulness. Because Ds elements preferentially transpose to nearby genomic sites, they can be used in targeted mutagenesis of linked genes. To efficiently target all genes throughout the genome, we generated a large population of transgenic Arabidopsis plants containing the Ds launch pad construct, identified lines containing single Ds launch pad inserts, and mapped the positions of Ds launch pads in 89 lines. The integration sites of the Ds launch pads were relatively evenly distributed on all five chromosomes, except for a region of chromosomes 2 and 4 and the centromeric regions. This resource therefore provides access to the majority of the Arabidopsis genome for targeted tagging.

RopGAP4-dependent Rop GTPase rheostat control of Arabidopsis oxygen deprivation tolerance.

Transient soil flooding limits cellular oxygen to roots and reduces crop yield. Plant response to oxygen deprivation involves increased expression of the alcohol dehydrogenase gene (ADH) and ethanolic fermentation. Disruption of the Arabidopsis gene that encodes Rop (RHO-like small G protein of plants) guanosine triphosphatase (GTPase) activating protein 4 (ROPGAP4), a Rop deactivator, elevates ADH expression in response to oxygen deprivation but decreases tolerance to stress. Rop-dependent production of hydrogen peroxide via a diphenylene iodonium chloride-sensitive calcium-dependent reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is necessary for induction of both ADH and RopGAP4 expression. Tolerance to oxygen deprivation requires Rop activation and RopGAP4-dependent negative feedback regulation. This Rop signal transduction rheostat balances the ability to increase ethanolic fermentation with survival.