Anna N. Stepanova

Five components of the ethylene-response pathway identified in a screen for weak ethylene-insensitive mutants in Arabidopsis

Five ethylene-insensitive loci (wei1–wei5) were identified by using a low-dose screen for “weak” ethylene-insensitive mutants. wei1, wei2, and wei3 seedlings showed hormone insensitivity only in roots, whereas wei4 and wei5 displayed insensitivity in both roots and hypocotyls. The genes corresponding to wei1, wei4, and wei5 were isolated using a positional cloning approach. The wei1 mutant harbored a recessive mutation in TIR1, which encodes a component of the SCF protein ubiquitin ligase involved in the auxin response. wei4, a dominant mutant, resulted from a mutation in the ethylene receptor ERS, whereas wei5, a semidominant mutant, was caused by a mutation in the EIN3-related transcription factor gene EIL1. The simultaneous loss of functional WEI5/EIL1 and EIN3 nearly completely abolished the ethylene response in etiolated seedlings, and adult plants were highly susceptible to infection by the necrotrophic fungal pathogen Botrytis cinerea. Moreover, wei5/eil1 ein3 double mutants were able to fully suppress constitutive signaling caused by ctr1, suggesting a synergistic interaction among these gene products. Unlike previously known root ethylene-insensitive mutants, wei2 and wei3 were not affected in their response to auxin and showed a normal response to gravity. Genetic mapping studies indicate that wei2 and wei3 correspond to previously unidentified ethylene pathway genes that may control cell-elongation processes functioning at the intersection of the ethylene and auxin response pathways.

Ethylene signaling and response: where different regulatory modules meet.

The structural simplicity of the gaseous hormone ethylene stands in contrast with the complexity of the physiological processes ethylene regulates. Initial studies suggested a simple linear arrangement of signaling molecules leading from the ethylene receptors to the EIN3 family of transcription factors. Recent discoveries have substantially changed this view. Current models suggest existence of a complex signaling pathway composed of several phosphorylation cascades, feedback-regulated transcriptional networks, and protein and mRNA turnover regulatory modules. Interactions between ethylene and other signals determine which of the ethylene-mediated responses get activated in a particular cell at a particular time. Tissue-specific regulation of auxin biosynthesis, transport, and response by ethylene is emerging as a key element in this signal integration process.

Ethylene signaling: simple ligand, complex regulation

The hormone ethylene plays numerous roles in plant development. In the last few years the model of ethylene signaling has evolved from an initially largely linear route to a much more complex pathway with multiple feedback loops. Identification of key transcriptional and post-transcriptional regulatory modules controlling expression and/or stability of the core pathway components revealed that ethylene perception and signaling are tightly regulated at multiple levels. This review describes the most current outlook on ethylene signal transduction and emphasizes the latest discoveries in the ethylene field that shed light on the mechanistic mode of action of the central pathway components CTR1 and EIN2, as well as on the post-transcriptional regulatory steps that modulate the signaling flow through the pathway.

Local auxin biosynthesis modulates gradient-directed planar polarity in Arabidopsis

The coordination of cell polarity within the plane of a single tissue layer (planar polarity) is a crucial task during development of multicellular organisms. Mechanisms underlying establishment of planar polarity, however, differ substantially between plants and animals. In Arabidopsis thaliana, planar polarity of root-hair positioning along epidermal cells is coordinated towards maximum concentration of an auxin gradient in the root tip. This gradient has been hypothesized to be sink-driven and computational modelling suggests that auxin efflux carrier activity may be sufficient to generate the gradient in the absence of auxin biosynthesis in the root. Here, we demonstrate that the Raf-like kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1; Refs 8, 9) acts as a concentration-dependent repressor of a biosynthesis-dependent auxin gradient that modulates planar polarity in the root tip. We analysed auxin biosynthesis and concentration gradients in a variety of root-hair-position mutants affected in CTR1 activity, auxin biosynthesis and transport. Our results reveal that planar polarity relies on influx- and efflux-carrier-mediated auxin redistribution from a local biosynthesis maximum. Thus, a local source of auxin biosynthesis contributes to gradient homeostasis during long-range coordination of cellular morphogenesis.

A Small-Molecule Screen Identifies l-Kynurenine as a Competitive Inhibitor of TAA1/TAR Activity in Ethylene-Directed Auxin Biosynthesis and Root Growth in Arabidopsis

In this work, Kyn is identified as an auxin biosynthesis inhibitor that effectively and selectively targets TAA1-like Trp aminotransferases. Moreover, it describes a previously undiscovered positive feedback loop between auxin biosynthesis and ethylene signaling pathways in roots. The interactions between phytohormones are crucial for plants to adapt to complex environmental changes. One example is the ethylene-regulated local auxin biosynthesis in roots, which partly contributes to ethylene-directed root development and gravitropism. Using a chemical biology approach, we identified a small molecule, l-kynurenine (Kyn), which effectively inhibited ethylene responses in Arabidopsis thaliana root tissues. Kyn application repressed nuclear accumulation of the ETHYLENE INSENSITIVE3 (EIN3) transcription factor. Moreover, Kyn application decreased ethylene-induced auxin biosynthesis in roots, and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE RELATEDs (TAA1/TARs), the key enzymes in the indole-3-pyruvic acid pathway of auxin biosynthesis, were identified as the molecular targets of Kyn. Further biochemical and phenotypic analyses revealed that Kyn, being an alternate substrate, competitively inhibits TAA1/TAR activity, and Kyn treatment mimicked the loss of TAA1/TAR functions. Molecular modeling and sequence alignments suggested that Kyn effectively and selectively binds to the substrate pocket of TAA1/TAR proteins but not those of other families of aminotransferases. To elucidate the destabilizing effect of Kyn on EIN3, we further found that auxin enhanced EIN3 nuclear accumulation in an EIN3 BINDING F-BOX PROTEIN1 (EBF1)/EBF2-dependent manner, suggesting the existence of a positive feedback loop between auxin biosynthesis and ethylene signaling. Thus, our study not only reveals a new level of interactions between ethylene and auxin pathways but also offers an efficient method to explore and exploit TAA1/TAR-dependent auxin biosynthesis.

Short-Term Growth Responses to Ethylene in Arabidopsis Seedlings Are EIN3/EIL1 Independent

Kinetic studies indicate there are two phases to growth inhibition by ethylene for the hypocotyls of etiolated Arabidopsis seedlings. Phase I is transient, while phase II results in sustained growth inhibition. The EIN2 membrane protein is required for both the first and second phases of growth inhibition by ethylene, while the transcription factors EIN3 and EIL1 are required for the second phase but not the first phase. The first phase lasts no more than 2 h. It is less sensitive to the ethylene response inhibitor 1-methylcyclopropene and more sensitive to ethylene than the second phase. The first phase shows adaptation at low concentrations of ethylene (≤0.01 μL L−1) with a relative refractory period of 5 h after ethylene is added. A modified signal transduction model is proposed that accounts for the two phases of growth inhibition.

Local Auxin Sources Orient the Apical-Basal Axis in Arabidopsis Embryos

Establishment of the embryonic axis foreshadows the main body axis of adults both in plants and in animals, but underlying mechanisms are considered distinct. Plants utilize directional, cell-to-cell transport of the growth hormone auxin to generate an asymmetric auxin response that specifies the embryonic apical-basal axis. The auxin flow directionality depends on the polarized subcellular localization of PIN-FORMED (PIN) auxin transporters. It remains unknown which mechanisms and spatial cues guide cell polarization and axis orientation in early embryos. Herein, we provide conceptually novel insights into the formation of embryonic axis in Arabidopsis by identifying a crucial role of localized tryptophan-dependent auxin biosynthesis. Local auxin production at the base of young embryos and the accompanying PIN7-mediated auxin flow toward the proembryo are required for the apical auxin response maximum and the specification of apical embryonic structures. Later in embryogenesis, the precisely timed onset of localized apical auxin biosynthesis mediates PIN1 polarization, basal auxin response maximum, and specification of the root pole. Thus, the tight spatiotemporal control of distinct local auxin sources provides a necessary, non-cell-autonomous trigger for the coordinated cell polarization and subsequent apical-basal axis orientation during embryogenesis and, presumably, also for other polarization events during postembryonic plant life.

T-DNA Mutagenesis in Arabidopsis

Insertional mutagenesis is a basic genetic tool that allows for a rapid identification of the tagged genes responsible for a particular phenotype. Transposon and Agrobacterium-mediated DNA integration are the most commonly used biological mutagens in plants. The main drawback of these technologies is the relatively low frequency of mutations, as compared to those induced by conventional chemical or physical agents, thus limiting the use of insertional mutagens to the generation of large mutant populations in few genetic backgrounds. Recent improvements in Agrobacterium-mediated transformation efficiency and an increasing repertoire of transformation vectors available to the research community is making this type of mutagen very attractive for individual laboratories interested in the studies of mutations in particular genetic backgrounds. Herein, we describe a simple yet robust Arabidopsis transformation procedure that can be used to generate large numbers of insertional mutants in Arabidopsis thaliana. Using this protocol, transformation efficiencies of up to 5% can be achieved.

A Recombineering-Based Gene Tagging System for Arabidopsis

One of the most information-rich aspects of gene functional studies is characterization of gene expression profiles at cellular resolution, and subcellular localization of the corresponding proteins. These studies require visualization of the endogenous gene products using specific antibodies, or, more commonly, generation of whole-gene translational fusions with a reporter gene such as a fluorescent protein. To facilitate the generation of such translational fusions and to ensure that all cis-regulatory sequences are included, we have used a bacterial homologous recombination system (recombineering) to insert fluorescent protein tags into genes of interest harbored by transformation-competent bacterial artificial chromosomes (TACs). This approach has several advantages compared to other classical strategies. First, the researcher does not have to guess what the regulatory sequences of a gene are, as tens of thousands of base pairs flanking the gene of interest can be included in the construct. Second, because the genes of interest are not amplified by PCR, there are practically no limits to the size of a gene that can be tagged. Third, there are no restrictions on the location in which the fluorescent protein can be inserted, as the position is determined by sequence homology with the recombination primers. Finally, all of the required strains and TAC clones are publically available, and the experimental procedures described here are simple and robust. Thus, we suggest that recombineering-based gene tagging should be the gold standard for gene expression studies in Arabidopsis.

Transcriptomic Signature of the SHATTERPROOF2 Expression Domain Reveals the Meristematic Nature of Arabidopsis Gynoecial Medial Domain

Transcriptional profiles of spatially and temporally restricted cell populations from the Arabidopsis gynoecium reveals the meristematic nature of the gynoecial medial domain. Plant meristems, like animal stem cell niches, maintain a pool of multipotent, undifferentiated cells that divide and differentiate to give rise to organs. In Arabidopsis (Arabidopsis thaliana), the carpel margin meristem is a vital meristematic structure that generates ovules from the medial domain of the gynoecium, the female floral reproductive structure. The molecular mechanisms that specify this meristematic region and regulate its organogenic potential are poorly understood. Here, we present a novel approach to analyze the transcriptional signature of the medial domain of the Arabidopsis gynoecium, highlighting the developmental stages that immediately proceed ovule initiation, the earliest stages of seed development. Using a floral synchronization system and a SHATTERPROOF2 (SHP2) domain-specific reporter, paired with FACS and RNA sequencing, we assayed the transcriptome of the gynoecial medial domain with temporal and spatial precision. This analysis reveals a set of genes that are differentially expressed within the SHP2 expression domain, including genes that have been shown previously to function during the development of medial domain-derived structures, including the ovules, thus validating our approach. Global analyses of the transcriptomic data set indicate a similarity of the pSHP2-expressing cell population to previously characterized meristematic domains, further supporting the meristematic nature of this gynoecial tissue. Our method identifies additional genes including novel isoforms, cis-natural antisense transcripts, and a previously unrecognized member of the REPRODUCTIVE MERISTEM family of transcriptional regulators that are potential novel regulators of medial domain development. This data set provides genome-wide transcriptional insight into the development of the carpel margin meristem in Arabidopsis.