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INTRODUCTION Pattern formation is one of four major developmental processes that generate the adult body in multicellular organisms. The other processes--growth, morphogenesis and differentiation--can readily be defined in terms of [...]

In dicotyledonous plants, primordia of most seedling organs are laiddown between fertilization and the formation of the heart-stage embryo. In Arabidopsis embryogenesis, highly regular cell divisions and cell expansions facilitate the characterization of mutant development.We have taken a genetic approach to identify genes involved in organising the hypocotyl/root axis. The initiation of this axis is marked by oriented expansions and longitudinal divisions of cells in the lower tier of the early globular embryo. These cells go on to form a defined number of parallel cell files that constitute the hypocotyl and most of the radicle. Mutants impaired in the initiation or elaboration of the hypocotyl/root axis were selected by their seedling phenotype and subsequently analysed at embryonic stages. Several conclusions are suggested by the phenotypes of these mutants. First, hypocotyl/root axis formation can be genetically separated from other aspects of embryonic pattern formation. Second, initiation of the hypocotyl/rootaxis appears to be genetically distinct from post-embryonic root initiation. Third, four loci were identified that appear to contribute to the elaboration of the axial pattern. Finally, an anatomical inspection of one of the mutants, amenable to an analysis at post-embryonicstages, suggests a genetic link between basal pattern formation in the embryo and aspects of vascular differentiation in the adult plant. [ABSTRACT FROM AUTHOR]

The regulatory effect auxin has on its own transport is critical in numerous self-organizing plant patterning processes. However, our understanding of the molecular mechanisms linking auxin signal transduction and auxin transport is still fragmentary, and important regulatory genes remain to be identified. To track a key link between auxin signaling and auxin transport in development, we established an Arabidopsis thaliana genetic background in which fundamental patterning processes in both shoot and root were essentially abolished and the expression of PIN FORMED (PIN) auxin efflux facilitators was dramatically reduced. In this background, we demonstrate that activating a steroid-inducible variant of the auxin response factor (ARF) MONOPTEROS (MP) is sufficient to restore patterning and PIN gene expression. Further, we show that MP binds to distinct promoter elements of multiple genetically defined PIN genes. Our work identifies a direct regulatory link between central, well-characterized genes involved in auxin signal transduction and auxin transport. The steroid-inducible MP system directly demonstrates the importance of this molecular link in multiple patterning events in embryos, shoots and roots, and provides novel options for interrogating the properties of self-regulated auxin-based patterning in planta., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

The current model of auxin-inducible transcription describes numerous regulatory interactions between AUXIN RESPONSE FACTORs (ARFs) and Aux/IAAs. However, specific relationships between individual members of these families in planta remain largely uncharacterized. Using a systems biology approach, the entire suite of Aux/IAA genes directly regulated by the developmentally pivotal ARF MONOPTEROS (MP) was recently determined for multiple Arabidopsis tissue types. This study showed that MP directly targets distinct subclades of Aux/IAAs, revealing potential regulatory modules of redundantly acting Aux/IAAs involved in MP-dependent processes. Further, functional analyses indicated that the protein products of these targeted Aux/IAAs negatively feedback on MP. Thus, comprehensive identification of Aux/IAAs targeted by individual ARFs will generate biologically meaningful networks of ARF-Aux/IAA regulatory modules controlling distinct plant pathways.

De novo meristem formation in tissue culture critically depends on the correct organization of hormonal domains, which is followed by expression shoot meristem pattern genes. The genetic basis of plant regeneration is fragmentary, but mutant studies demonstrate that signaling through MONOPTEROS (MP)/AUXIN RESPONSE FACTOR 5 is necessary for the formation of shoots from Arabidopsis calli. Most strikingly, variants of MP, uncoupling MP activity from negative regulation by Aux/IAA proteins, showed that MP is also sufficient for promoting de novo shoot formation even from normally recalcitrant tissues. In this function MP acts through pathways involving the homeobox transcription factor SHOOT MERISTEMLESS (STM) and AP2 domain transcription factor CYTOKININ RESPONSE FACTOR2 (CRF2). Our findings provide an entry point to better address the molecular genetics underlying divergent regeneration properties and demonstrate the potential of ARF-derived constructs as novel genetic tools to develop high frequency regeneration systems in recalcitrant explants and species.

In vitro regeneration of complete organisms from diverse cell types is a spectacular property of plant cells. Despite the great importance of plant regeneration for plant breeding and biotechnology, its molecular basis is still largely unclear and many important crop plants have remained recalcitrant to regeneration. Hormone-exposure protocols to trigger the de novo formation of either roots or shoots from callus tissue demonstrate the importance of auxin and cytokinin signaling pathways, and genetic differences in these pathways may contribute to the highly divergent responsiveness of plant species to regeneration protocols. In this study, we show that signaling through MONOPTEROS (MP)/AUXIN RESPONSE FACTOR 5 is necessary for the formation of shoots from Arabidopsis calli. Most strikingly, an irrepressible variant of MP, MPΔ, is sufficient for promoting de novo shoot formation through pathways involving the genetically downstream functions of SHOOT MERISTEMLESS (STM) and CYTOKININ RESPONSE FACTOR2 (CRF2). We conclude that the MPΔ genotype can promote de novo shoot formation and can be used to probe corresponding signaling pathways., (© 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.)

The regulatory interactions between AUXIN RESPONSE FACTORS (ARFs) and Aux/IAA repressors play a central role in auxin signal transduction. Yet, the systems properties of this regulatory network are not well established. We generated a steroid-inducible ARF5/MONOPTEROS (MP) transgenic background to survey the involvement of this factor in the transcriptional regulation of the entire Aux/IAA family in Arabidopsis thaliana. Target genes of ARF5/MP identified by this approach were confirmed by chromatin immunoprecipitation, in vitro gel retardation assays and gene expression analyses. Our study shows that ARF5/MP is indispensable for the correct regulation of nearly one-half of all Aux/IAA genes, and that these targets coincide with distinct subclades. Further, genetic analyses demonstrate that the protein products of multiple Aux/IAA targets negatively feed back onto ARF5/MP activity. This work indicates that ARF5/MP broadly influences the expression of the Aux/IAA gene family, and suggests that such regulation involves the activation of specific subsets of redundantly functioning factors. These groups of factors may then act together to control various processes within the plant through negative feedback on ARF5. Similar detailed analyses of other Aux/IAA-ARF regulatory modules will be required to fully understand how auxin signal transduction influences virtually every aspect of plant growth and development., (© 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.)

In Arabidopsis thaliana, the HUA2 gene is required for proper expression of FLOWERING LOCUS C (FLC) and AGAMOUS, key regulators of flowering time and reproductive development, respectively. Although HUA2 is broadly expressed, plants lacking HUA2 function have only moderately reduced plant stature, leaf initiation rate and flowering time. To better understand HUA2 activity, and to test whether redundancy with similar genes underlies the absence of strong phenotypes in HUA2 mutant plants, we identified and subsequently characterized three additional HUA2-LIKE (HULK) genes in Arabidopsis. These genes form two clades (HUA2/HULK1 and HULK2/HULK3), with members broadly conserved in both vascular and non-vascular plants, but not present outside the plant kingdom. Plants with progressively reduced HULK activity had increasingly severe developmental defects, and plants homozygous for loss-of-function mutations in all four HULK genes were not recovered. Multiple mutants displayed reproductive, embryonic and post-embryonic abnormalities, and provide detailed insights into the overlapping and unique functions of individual HULK genes. With regard to flowering time, opposing influences were apparent: hua2 hulk1 plants were early-flowering, while hulk2 hulk3 mutants were late-flowering, and hua2 acted epistatically to cause early flowering in all combinations. Genome-wide expression profiling of mutant combinations using RNA-Seq revealed complex transcriptional changes in seedlings, with FLC, a known target of HUA2, among the most affected. Our studies, which include characterization of HULK expression patterns and subcellular localization, suggest that the HULK genes encode conserved nuclear factors with partially redundant but essential functions associated with diverse genetic pathways in plants., (© 2014 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Self-regulatory patterning mechanisms capable of generating biologically meaningful, yet unpredictable cellular patterns offer unique opportunities for obtaining mathematical descriptions of underlying patterning systems properties. The networks of higher-order veins in leaf primordia constitute such a self-regulatory system. During the formation of higher-order veins, vascular precursors are selected from a homogenous field of subepidermal cells in unpredictable positions to eventually connect in complex cellular networks. Auxin transport routes have been implicated in this selection process, but understanding of their role in vascular patterning has been limited by our inability to monitor early auxin transport dynamics in vivo. Here we describe a live-imaging system in emerging Arabidopsis thaliana leaves that uses a PIN1:GFP reporter to visualize auxin transport routes and an Athb8:YFP reporter as a marker for vascular commitment. Live-imaging revealed common features initiating the formation of all higher-order veins. The formation of broad PIN1 expression domains is followed by their restriction, leading to sustained, elevated PIN1 expression in incipient procambial cells files, which then express Athb8. Higher-order PIN1 expression domains (hPEDs) are initiated as freely ending domains that extend toward each other and sometimes fuse with them, creating connected domains. During the restriction and specification phase, cells in wider hPEDs are partitioned into vascular and non-vascular fates: Central cells acquire a coordinated cell axis and express elevated PIN1 levels as well as the pre-procambial marker Athb8, while edge cells downregulate PIN1 and remain isodiametric. The dynamic nature of the early selection process is underscored by the instability of early hPEDs, which can result in dramatic changes in vascular network architecture prior to Athb8 expression, which is correlated with the promotion onto vascular cell fate.

This study describes the generation and test of a genetic resource suited to identify determinants of cell biological traits in plants. The use of quantitative trait loci (QTL) mapping for a better genetic understanding of cell biological traits is still at an early stage, even for biotechnologically important cell properties, such as the dimensions of fiber cells. A common strategy, the mapping of QTLs in recombinant inbred line (RIL) populations, is limited by the fact that the existing RIL populations exploit only a small fraction of the existing natural variation. Here, we report the mapping of QTLs impacting on the length of fiber cells in Arabidopsis inflorescence stems in a newly generated RIL population derived from a cross between the accessions Berkeley and the little known Lz-0. Through inbreeding of individual F(2) plants, a total of 159 new F8 lines were produced and genotyped with a set of 49 single nucleotide polymorphism markers. The population was successfully used not only for the mapping of three QTLs controlling fiber length, but also to map five QTL controlling flowering time under short and long-day conditions. Our study demonstrates the usefulness of this new genetic resource by mapping in it QTLs underlying a poorly explored cellular trait as well as an already better explored regulatory pathway. The new RIL population and an online platform for the continuous supplementation of genetic markers will be generally available to substantially broaden the genetic diversity through which loci with impact on plant quantitative traits can be identified.

Fibre properties and the biochemical composition of cell walls are important traits in many applications. For example, the lengths of fibres define the strength and quality of paper, and lignin content is a critical parameter for the use of biomass in biofuel production. Identifying genes controlling these traits is comparatively difficult in woody species, because of long generation times and limited amenability to high-resolution genetic mapping. To address this problem, this study mapped quantitative trait loci (QTLs) defining fibre length and lignin content in the Arabidopsis recombinant inbred line population Col-4 × Ler-0. Adapting high-throughput phenotyping techniques for both traits for measurements in Arabidopsis inflorescence stems identified significant QTLs for fibre length on chromosomes 2 and 5, as well as one significant QTL affecting lignin content on chromosome 2. For fibre length, total variation within the population was 208% higher than between parental lines and the identified QTLs explained 50.58% of the observed variation. For lignin content, the values were 261 and 26.51%, respectively. Bioinformatics analysis of the associated intervals identified a number of candidate genes for fibre length and lignin content. This study demonstrates that molecular mapping of QTLs pertaining to wood and fibre properties is possible in Arabidopsis, which substantially broadens the use of Arabidopsis as a model species for the functional characterization of plant genes.

The molecularly well-characterized auxin signal transduction pathway involves two evolutionarily conserved families interacting through their C-terminal domains III and IV: the Auxin Response Factors (ARFs) and their repressors the Aux/IAAs, to control auxin-responsive genes, among them genes involved in auxin transport. ( 1) (,) ( 2) We have developed a new genetic tool to study ARF function. Using MONOPTEROS (MP)/ARF5, we have generated a truncated version of MP (MPΔ), ( 3) which has lost the target domains for repression by Aux/IAA proteins. Besides exploring genetic interactions between MP and Aux/IAAs, we used this construct to trace MP's role in vascular patterning, a previously characterized auxin dependent process. ( 4) (,) ( 5) Here we summarize examples of naturally occurring truncated ARFs and summarize potential applications of truncated ARFs as analytical tools.

The establishment of adaxial-abaxial polarity in plant lateral organs involves elaborate interactions between members of several transcription factor families, including the Auxin Response Factors (ARFs). We previously described a dominant allele of ARF5/MONOPTEROS (MP), termed MPΔ, which causes severe vascular hypertrophy in shoot lateral organs. Here we report that these organs are also disrupted in adaxial-abaxial polarity. Other MPΔ lateral organs with decreased vasculature show similar disruptions, suggesting that MP impinges on organ polarity through pathways separate from its role in promoting vascularization. Furthermore, we demonstrate that MPΔ exhibits an adaxial-abaxial asymmetry in its ability to influence organ development. Since ARFs previously implicated in polarity establishment function as transcriptional repressors, the transcriptional activator MP represents a novel link between auxin signal transduction and adaxial-abaxial polarity.

Combinatorial interactions of AUXIN RESPONSE FACTORs (ARFs) and auxin/indole acetic acid (Aux/IAA) proteins through their common domains III and IV regulate auxin responses, but insight into the functions of individual proteins is still limited. As a new tool to explore this regulatory network, we generated a gain-of-function ARF genotype by eliminating domains III and IV from the functionally well-characterized ARF MONOPTEROS(MP)/ARF5. This truncated version of MP, termed MPΔ, conferred complementing MP activity, but also displayed a number of semi-dominant traits affecting auxin signaling and organ patterning. In MPΔ, the expression levels of many auxin-inducible genes, as well as rooting properties and vascular tissue abundance, were enhanced. Lateral organs were narrow, pointed and filled with parallel veins. This effect was epistatic over the vascular hypotrophy imposed by certain Aux/IAA mutations. Further, in MPΔ leaves, failure to turn off the procambium-selecting gene PIN1 led to the early establishment of oversized central procambial domains and very limited subsequent lateral growth of the leaf lamina. We conclude that MPΔ can selectively uncouple a single ARF from regulation by Aux/IAA proteins and can be used as a genetic tool to probe auxin pathways and explore leaf development., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

The shoot and root apical meristems (SAM and RAM) formed during embryogenesis are crucial for postembryonic plant development. We report the identification of POPCORN (PCN), a gene required for embryo development and meristem organization in Arabidopsis thaliana. Map-based cloning revealed that PCN encodes a WD-40 protein expressed both during embryo development and postembryonically in the SAM and RAM. The two pcn alleles identified in this study are temperature sensitive, showing defective embryo development when grown at 22°C that is rescued when grown at 29°C. In pcn mutants, meristem-specific expression of WUSCHEL (WUS), CLAVATA3, and WUSCHEL-RELATED HOMEOBOX5 is not maintained; SHOOTMERISTEMLESS, BODENLOS (BDL) and MONOPTEROS (MP) are misexpressed. Several findings link PCN to auxin signaling and meristem function: ectopic expression of DR5(rev):green fluorescent protein (GFP), pBDL:BDL-GFP, and pMP:MP-β-glucuronidase in the meristem; altered polarity and expression of pPIN1:PIN1-GFP in the apical domain of the developing embryo; and resistance to auxin in the pcn mutants. The bdl mutation rescued embryo lethality of pcn, suggesting that improper auxin response is involved in pcn defects. Furthermore, WUS, PINFORMED1, PINOID, and TOPLESS are dosage sensitive in pcn, suggesting functional interaction. Together, our results suggest that PCN functions in the auxin pathway, integrating auxin signaling in the organization and maintenance of the SAM and RAM.

Since the hallmark discovery of Aequorea victoria's Green Fluorescent Protein (GFP) and its adaptation for efficient use in plants, fluorescent protein tags marking expression profiles or genuine proteins of interest have been used to recognize plant tissues and cell types, to monitor dynamic cell fate selection processes, and to obtain cell type-specific transcriptomes. Fluorescent tagging enabled visualization in living tissues and the precise recordings of dynamic expression pattern changes. The resulting accurate recording of cell fate acquisition kinetics in space and time has strongly stimulated mathematical modeling of self-organizing feedback mechanisms. In developmental studies, the use of fluorescent proteins has become critical, where morphological markers of tissues, cell types, or differentiation stages are either not known or not easily recognizable. In this review, we focus on the use of fluorescent markers to identify and illuminate otherwise invisible cell states in plant development.

Genes expressed in vascular tissues have been identified by several strategies, usually with a focus on mature vascular cells. In this study, we explored the possibility of using two opposite types of altered tissue compositions in combination with a double-filter selection to identify genes with a high probability of vascular expression in early organ primordia. Specifically, we generated full-transcriptome microarray profiles of plants with (a) genetically strongly reduced and (b) pharmacologically vastly increased vascular tissues and identified a reproducible cohort of 158 transcripts that fulfilled the dual requirement of being underrepresented in (a) and overrepresented in (b). In order to assess the predictive value of our identification scheme for vascular gene expression, we determined the expression patterns of genes in two unbiased subsamples. First, we assessed the expression patterns of all twenty annotated transcription factor genes from the cohort of 158 genes and found that seventeen of the twenty genes were preferentially expressed in leaf vascular cells. Remarkably, fifteen of these seventeen vascular genes were clearly expressed already very early in leaf vein development. Twelve genes with published leaf expression patterns served as a second subsample to monitor the representation of vascular genes in our cohort. Of those twelve genes, eleven were preferentially expressed in leaf vascular tissues. Based on these results we propose that our compendium of 158 genes represents a sample that is highly enriched for genes expressed in vascular tissues and that our approach is particularly suited to detect genes expressed in vascular cell lineages at early stages of their inception., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

A central question in developmental biology is how multicellular organisms coordinate cell division and differentiation to determine organ size. In Arabidopsis roots, this balance is controlled by cytokinin-induced expression of SHORT HYPOCOTYL 2 (SHY2) in the so-called transition zone of the meristem, where SHY2 negatively regulates auxin response factors (ARFs) by protein-protein interaction. The resulting down-regulation of PIN-FORMED (PIN) auxin efflux carriers is considered the key event in promoting differentiation of meristematic cells. Here we show that this regulation involves additional, intermediary factors and is spatio-temporally constrained. We found that the described cytokinin-auxin crosstalk antagonizes BREVIS RADIX (BRX) activity in the developing protophloem. BRX is an auxin-responsive target of the prototypical ARF MONOPTEROS (MP), a key promoter of vascular development, and transiently enhances PIN3 expression to promote meristem growth in young roots. At later stages, cytokinin induction of SHY2 in the vascular transition zone restricts BRX expression to down-regulate PIN3 and thus limit meristem growth. Interestingly, proper SHY2 expression requires BRX, which could reflect feedback on the auxin responsiveness of SHY2 because BRX protein can directly interact with MP, likely acting as a cofactor. Thus, cross-regulatory antagonism between BRX and SHY2 could determine ARF activity in the protophloem. Our data suggest a model in which the regulatory interactions favor BRX expression in the early proximal meristem and SHY2 prevails because of supplementary cytokinin induction in the later distal meristem. The complex equilibrium of this regulatory module might represent a universal switch in the transition toward differentiation in various developmental contexts.

The analytical power of Arabidopsis thaliana genomics has turned its local varieties (accessions) from divergent habitats into important genetic resources. Variant alleles harbored in those accessions are used to identify loci controlling important plant traits with enormous benefits for analytical as well as applied purposes. We argue here that the information derived from Arabidopsis accessions can be further expanded, if a systematic effort for recording the growth conditions of new Arabidopsis accessions is rapidly implemented. The modest and feasible changes in genetic sampling practice that we propose will dramatically increase the quality and quantity of data obtained from Arabidopsis accessions. The broader data set will no longer focus solely on the genetic mechanism within the plant, but will also address the plant's interaction with its environment. We suggest (a) a modified sampling strategy involving sample size and the recording of additional growth conditions (Appendix) and (b) the establishment of a centralized and expandable database to cover all available information regarding the habitats of Arabidopsis accessions.

Cell type-specific fluorescent gene expression markers are a prerequisite for various strategies in functional genomics and developmental biology. To increase the resolution of vascular tissue analysis and to identify more genes expressed in the vasculature, we searched for expression in vascular tissues within a new collection of transactivated enhancer trap lines. Among 33 lines with vascular expression, we identified five lines with expression profiles marking procambial or procambium-associated cell states. In stem cross-sections we identified one line with phloem- and four with xylem-specific expression, as well as nine lines with expression in both phloem and xylem and two with cambial expression. For all lines, we also report the expression patterns in different organs and developmental stages. Special features of expression patterns include a line with auxin-dependent expression domains. We determined the flanking sequences of 21 enhancer trap insertions, 16 of which are found in, or in close proximity to, annotated genes and thus may reflect the expression patterns of natural promoters. Finally, we analyzed the loss-of-function phenotypes of 14 putatively affected genes. Remarkably, mutations in a gene encoding a putative F-box protein were associated with an auxin-hypersensitive hypocotyl elongation response. Our compendium provides a diverse selection of markers for different vascular cell states, which can be used for targeted gene expression, cell type-specific transcript profiling and gene function assignment in the plant vascular system.

During embryogenesis a single cell gives rise to a functional multicellular organism. In higher plants, as in many other multicellular systems, essential architectural features, such as body axes and major tissue layers are established early in embryogenesis and serve as a positional framework for subsequent pattern elaboration. In Arabidopsis, the apicalbasal axis and the radial pattern of tissues wrapped around it are already recognizable in young embryos of only about a hundred cells in size. This early axial pattern seems to provide a coordinate system for the embryonic initiation of shoot and root. Findings from genetic studies in Arabidopsis are revealing molecular mechanisms underlying the initial establishment of the axial core pattern and its subsequent elaboration into functional shoots and roots. The genetic programs operating in the early embryo organize functional cell patterns rapidly and reproducibly from minimal cell numbers. Understanding their molecular details could therefore greatly expand our ability to generate plant body patterns de novo, with important implications for plant breeding and biotechnology.

The phytohormone auxin plays a pivotal role in plant development, regulating a myriad of processes including embryo patterning, root patterning, organ initiation, and vein patterning. Auxin is unique among the plant hormones as it is actively transported from cell to cell in a polar fashion. It has recently been discovered that polar auxin transport generates dynamic, local auxin gradients within plant tissues that appear to provide positional information in patterning processes. Visualization of apparent auxin transport patterns has largely been facilitated by the recent creation of translational fusions of GFP to members of the Arabidopsis (At)PIN family of auxin efflux associated proteins. Confocal visualization of these fusion products (PIN:GFPs) enables the tracking of apparent auxin transport patterns in a huge number of samples. This visualization method can be combined with experimental interference, such as local auxin application and inhibition of auxin transport, to deduce possible self-organizing auxin-dependent patterning mechanisms and to make them amenable to mathematical modeling.

Initiation of leaves at the flanks of the shoot apical meristem occurs at sites of auxin accumulation and pronounced expression of auxin-inducible PIN-FORMED1 (PIN) genes, suggesting a feedback loop to progressively focus auxin in concrete spots. Because PIN expression is regulated by auxin response factor activity, including MONOPTEROS (MP), it appeared possible that MP affects leaf formation as a positive regulator of PIN genes and auxin transport. Here, we analyze a novel, completely leafless phenotype arising from simultaneous interference with both auxin signaling and auxin transport. We show that mp pin1 double mutants, as well as mp mutants treated with auxin-efflux inhibitors, display synergistic abnormalities not seen in wild type regardless of how strongly auxin transport was reduced. The synergism of abnormalities indicates that the role of MP in shoot meristem organization is not limited to auxin transport regulation. In the mp mutant background, auxin transport inhibition completely abolishes leaf formation. Instead of forming leaves, the abnormal shoot meristems dramatically increase in size, harboring correspondingly enlarged expression domains of CLAVATA3 and SHOOTMERISTEMLESS, molecular markers for the central stem cell zone and the complete meristem, respectively. The observed synergism under conditions of auxin efflux inhibition was further supported by an unrestricted PIN1 expression in mp meristems, as compared to a partial restriction in wild-type meristems. Auxin transport-inhibited mp meristems also lacked detectable auxin maxima. We conclude that MP promotes the focusing of auxin and leaf initiation in part through pathways not affected by auxin efflux inhibitors.

The acetyl bromide method has been modified to enable the rapid microscale determination of lignin content in Arabidopsis with the goal of determining the genes that control lignin in plants. Modifications include reduction in sample size, use of a microball mill, adoption of a modified rapid method of extraction, use of an ice-bath to stabilize solutions and reduction in the volume of solutions. The microscale method was shown to be rapid, accurate and precise with values in agreement with those determined by the full-scale acetyl bromide method. The extinction coefficient for Arabidopsis lignin, dissolved using acetyl bromide, was determined to be 23.35 g(-1) L cm(-1) at 280 nm. This value is independent of the Arabidopsis accession, environmental growth conditions and is insensitive to lignin structure. The newly developed method can be used to determine lignin content in the inflorescence stems of Arabidopsis for mapping of lignin-related genes.

AUXIN RESPONSE FACTOR (ARF)-mediated signaling conveys positional information during embryonic and postembryonic organogenesis and mutations in MONOPTEROS (MP/ARF5) result in severe patterning defects during embryonic and postembryonic development. Here we show that MP patterning activity is largely dispensable when the presumptive carboxypeptidase ALTERED MERISTEM PROGRAM 1 (AMP1) is not functional, indicating that MP is primarily necessary to counteract AMP1 activity. Closer inspection of the single and double mutant phenotypes reveals antagonistic influences of both genes on meristematic activities throughout the Arabidopsis life cycle. In the absence of MP activity, cells in apical meristems and along the paths of procambium formation acquire differentiated identities and this is largely dependent on differentiation-promoting AMP1 activity. Positions of antagonistic interaction between MP and AMP1 coincide with MP expression domains within the larger AMP1 expression domain. These observations suggest a model in which auxin-derived positional information through MP carves out meristematic niches by locally overcoming a general differentiation-promoting activity involving AMP1.

Polar auxin transport intimately connects plant cell polarity and multicellular patterning. Through the transport of the small molecule indole-3-acetic acid, plant cells integrate their polarities and communicate the degree of their polarization. In this way, they generate an apical-basal axis that serves as a positional reference anchoring subsequent patterning events. Research in recent years has brought the molecular mechanisms underlying auxin perception and auxin transport to light. This knowledge has been used to derive spectacular molecular visualization tools and animated computer simulations, which are now allied in a joint systems biology effort towards a mathematical description of auxin-transport-mediated patterning processes.

The formation of the leaf vascular pattern has fascinated biologists for centuries. In the early leaf primordium, complex networks of procambial cells emerge from homogeneous subepidermal tissue. The molecular nature of the underlying positional information is unknown, but various lines of evidence implicate gradually restricted transport routes of the plant hormone auxin in defining sites of procambium formation. Here we show that a crucial member of the AtPIN family of auxin-efflux-associated proteins, AtPIN1, is expressed prior to pre-procambial and procambial cell fate markers in domains that become restricted toward sites of procambium formation. Subcellular AtPIN1 polarity indicates that auxin is directed to distinct "convergence points" in the epidermis, from where it defines the positions of major veins. Integrated polarities in all emerging veins indicate auxin drainage toward pre-existing veins, but veins display divergent polarities as they become connected at both ends. Auxin application and transport inhibition reveal that convergence point positioning and AtPIN1 expression domain dynamics are self-organizing, auxin-transport-dependent processes. We derive a model for self-regulated, reiterative patterning of all vein orders and postulate at its onset a common epidermal auxin-focusing mechanism for major-vein positioning and phyllotactic patterning.

The extremely wide spectrum of the plant processes that are influenced by auxin raises the question of how signals conveyed by a single molecule can trigger such a variety of responses. Although many aspects of auxin function remain elusive, others have become genetically tractable. The identification of crucial genes in auxin signal transduction and auxin transport in the past few years has led to molecularly testable concepts of how auxin signals regulate gene activities in individual cells, and how the polar transport of auxin could impact on patterning processes throughout the plant.

During leaf development, ground meristem cells along continuous lines undergo coordinated oriented cell divisions and differentiate to form procambial cells, the precursors of all vascular cells. The molecular genetic dissection of early procambial development suffers from the lack of easily identifiable markers, especially of cell states preceding procambium formation. In this study, we have identified and characterized three reporter gene expression markers that reflect three distinct preprocambial stages, as well as one marker whose expression seems to be perfectly congruent with the appearance of procambial cells. All four markers are invariably expressed in continuous domains connected to pre-existing vasculature and their expression profiles reveal a common spatiotemporal pattern of early vein formation. We observed progressive extension of vascular strands at the preprocambial stage, suggesting that veins are initiated as freely ending preprocambial domains and that network formation occurs through subsequent fusion of these domains. Consistent with this interpretation, we demonstrate that veins are generally not programmed to become freely ending or interconnected network elements. Instead, we found that the progressive extension of preprocambial domains can be interrupted experimentally and that this leads to less complex vein patterns consisting of fewer vein orders, in which even lower-order veins become freely ending. Mesophyll differentiation turned out to be strictly correlated with the termination of preprocambial domain extension. These findings suggest that Arabidopsis vein pattern is not inherently determinate, but arises through reiterative initiation of new preprocambial branches until this process becomes terminated by the differentiation of mesophyll.

Transcription factors of the auxin response factor (ARF) family have been implicated in auxin-dependent gene regulation, but little is known about the functions of individual ARFs in plants. Here, interaction assays, expression studies and combinations of multiple loss- and gain-of-function mutants were used to assess the roles of two ARFs, NONPHOTOTROPIC HYPOCOTYL 4 (NPH4/ARF7) and MONOPTEROS (MP/ARF5), in Arabidopsis development. Both MP and NPH4 interact strongly and selectively with themselves and with each other, and are expressed in vastly overlapping domains. We show that the regulatory properties of both genes are far more related than suggested by their single mutant phenotypes. NPH4 and MP are capable of controlling both axis formation in the embryo and auxin-dependent cell expansion. Interaction of MP and NPH4 in Arabidopsis plants is indicated by their joint requirement in a number of auxin responses and by synergistic effects associated with the co-overexpression of both genes. Finally, we demonstrate antagonistic interaction between ARF and Aux/IAA gene functions in Arabidopsis development. Overexpression of MP suppresses numerous defects associated with a gain-of-function mutation in BODENLOS (BDL)/IAA12. Together these results provide evidence for the biological relevance of ARF-ARF and ARF-Aux/IAA interaction in Arabidopsis plants and demonstrate that an individual ARF can act in both invariantly programmed pattern formation as well as in conditional responses to external signals.

A number of observations have implicated auxin in the formation of vascular tissues in plant organs. These include vascular strand formation in response to local auxin application, the effects of impaired auxin transport on vascular patterns and suggestive phenotypes of Arabidopsis auxin response mutants. In this study, we have used molecular markers to visualize auxin response patterns in developing Arabidopsis leaves as well as Arabidopsis mutants and transgenic plants to trace pathways of auxin signal transduction controlling the expression of early procambial genes. We show that in young Arabidopsis leaf primordia, molecular auxin response patterns presage sites of procambial differentiation. This is the case not only in normal development but also upon experimental manipulation of auxin transport suggesting that local auxin signals are instrumental in patterning Arabidopsis leaf vasculature. We further found that the activity of the Arabidopsis gene MONOPTEROS, which is required for proper vascular differentiation, is also essential in a spectrum of auxin responses, which include the regulation of rapidly auxin-inducible AUX/IAA genes, and discovered the tissue-specific vascular expression profile of the class I homeodomain-leucine zipper gene, AtHB20. Interestingly, MONOPTEROS activity is a limiting factor in the expression of AtHB8 and AtHB20, two genes encoding transcriptional regulators expressed early in procambial development. Our observations connect general auxin signaling with early controls of vascular differentiation and suggest molecular mechanisms for auxin signaling in patterned cell differentiation.

The products of the cellulose synthase A (CESA) gene family are thought to function as isoforms of the cellulose synthase catalytic subunit, but for most CESA genes, the exact role in plant growth is still unknown. Assessing the function of individual CESA genes will require the identification of the null-mutant phenotypes and of the gene expression profiles for each gene. Here, we report that only four of 10 CESA genes, CESA1, CESA2, CESA3, and CESA9 are significantly expressed in the Arabidopsis embryo. We further identified two new mutations in the RADIALLY SWOLLEN1 (RSW1/CESA1) gene of Arabidopsis that obstruct organized growth in both shoot and root and interfere with cell division and cell expansion already in embryogenesis. One mutation is expected to completely abolish the enzymatic activity of RSW1(CESA1) because it eliminated one of three conserved Asp residues, which are considered essential for beta-glycosyltransferase activity. In this presumed null mutant, primary cell walls are still being formed, but are thin, highly undulated, and frequently interrupted. From the heart-stage onward, cell elongation in the embryo axis is severely impaired, and cell width is disproportionally increased. In the embryo, CESA1, CESA2, CESA3, and CESA9 are expressed in largely overlapping domains and may act cooperatively in higher order complexes. The embryonic phenotype of the presumed rsw1 null mutant indicates that the RSW1(CESA1) product has a critical, nonredundant function, but is nevertheless not strictly required for primary cell wall formation.

The overall morphology of a plant is largely determined by developmental decisions taken within or near the terminally positioned apical meristems of shoots and roots. The spatial separation of these developmental centers emphasizes the need for long-distance signaling. The same signaling events may simultaneously coordinate differentiation within meristems and in the connecting vascular tissues. Recent genetic and molecular analyses not only confirm the proposed role of auxin as a coordinating signal across the plant, but also implicate auxin as a patterning signal in embryo and meristem organization.

The plant hormone auxin has been implicated in vascular development, but the molecular details of patterned vascular differentiation have remained elusive. Research in the past year has identified new genes that control vascular patterning, and auxin transport and perception. New experimental strategies have been employed to study vascular development. Together, these findings have generated a conceptual framework and experimental tools for the exploration of vascular-tissue patterning at the molecular level.

How the complex patterns of plant vascular systems are generated is largely unknown. Advances in understanding vascular pattern formation at various levels are likely to follow recent large-scale genetic screens for Arabodopsis mutants with abnormal vascular systems.

The plant hormone auxin is central in the regulation of growth and development, however, the molecular basis for its action has remained enigmatic. In the absence of a molecular model, the wide range of responses elicited by auxin have been difficult to explain. Recent advances using molecular genetic approaches in Arabidopsis have led to the isolation of a number of key genes involved in auxin action. Of particular importance are genes involved in channelling polar auxin transport through the plant. In addition a model for auxin signal transduction, centred on regulated protein degradation, has been developed., (Copyright 1999 Academic Press.)