The auxin response factor MONOPTEROS controls meristem function and organogenesis in both the shoot and root through the direct regulation of PIN genes.

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.

Overcoming recalcitrance - Auxin response factor functions in plant regeneration.

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.

The identification and characterization of specific ARF-Aux/IAA regulatory modules in plant growth and development.

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.

Irrepressible MONOPTEROS/ARF5 promotes de novo shoot formation.

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.

Distinct subclades of Aux/IAA genes are direct targets of ARF5/MP transcriptional regulation.

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.

A plant-specific HUA2-LIKE (HULK) gene family in Arabidopsis thaliana is essential for development.

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.

Dynamic auxin transport patterns preceding vein formation revealed by live-imaging of Arabidopsis leaf primordia.

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.

Lz-0 × Berkeley: a new Arabidopsis recombinant inbred line population for the mapping of complex traits.

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.

Identification of quantitative trait loci controlling fibre length and lignin content in Arabidopsis thaliana stems.

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.

Irrepressible, truncated auxin response factors: natural roles and applications in dissecting auxin gene regulation pathways.

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.

A dominant mutation reveals asymmetry in MP/ARF5 function along the adaxial-abaxial axis of shoot lateral organs.

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.

Deletion of MP/ARF5 domains III and IV reveals a requirement for Aux/IAA regulation in Arabidopsis leaf vascular patterning.

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.

POPCORN functions in the auxin pathway to regulate embryonic body plan and meristem organization in Arabidopsis.

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-ß-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.

Glow in the dark: fluorescent proteins as cell and tissue-specific markers in plants.

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.

Double-filter identification of vascular-expressed genes using Arabidopsis plants with vascular hypertrophy and hypotrophy.

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.

Spatio-temporal sequence of cross-regulatory events in root meristem growth.

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.

Expanding ecological and evolutionary insights from wild Arabidopsis thaliana accessions.

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.

Tissue-specific GAL4 expression patterns as a resource enabling targeted gene expression, cell type-specific transcript profiling and gene function characterization in the Arabidopsis vascular system.

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.

Visualizing auxin transport routes in Arabidopsis leaf primordia.

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.

Embryogenesis: pattern formation from a single cell.

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.