Auxin-Regulated Reversible Inhibition of TMK1 Signaling by MAKR2 Modulates the Dynamics of Root Gravitropism.

Plants are able to orient their growth according to gravity, which ultimately controls both shoot and root architecture.1 Gravitropism is a dynamic process whereby gravistimulation induces the asymmetric distribution of the plant hormone auxin, leading to asymmetric growth, organ bending, and subsequent reset of auxin distribution back to the original pre-gravistimulation situation.1-3 Differential auxin accumulation during the gravitropic response depends on the activity of polarly localized PIN-FORMED (PIN) auxin-efflux carriers.1-4 In particular, the timing of this dynamic response is regulated by PIN2,5,6 but the underlying molecular mechanisms are poorly understood. Here, we show that MEMBRANE ASSOCIATED KINASE REGULATOR2 (MAKR2) controls the pace of the root gravitropic response. We found that MAKR2 is required for the PIN2 asymmetry during gravitropism by acting as a negative regulator of the cell-surface signaling mediated by the receptor-like kinase TRANSMEMBRANE KINASE1 (TMK1).2,7-10 Furthermore, we show that the MAKR2 inhibitory effect on TMK1 signaling is antagonized by auxin itself, which triggers rapid MAKR2 membrane dissociation in a TMK1-dependent manner. Our findings suggest that the timing of the root gravitropic response is orchestrated by the reversible inhibition of the TMK1 signaling pathway at the cell surface.

Framework for gradual progression of cell ontogeny in the Arabidopsis root meristem.

In plants, apical meristems allow continuous growth along the body axis. Within the root apical meristem, a group of slowly dividing quiescent center cells is thought to limit stem cell activity to directly neighboring cells, thus endowing them with unique properties, distinct from displaced daughters. This binary identity of the stem cells stands in apparent contradiction to the more gradual changes in cell division potential and differentiation that occur as cells move further away from the quiescent center. To address this paradox and to infer molecular organization of the root meristem, we used a whole-genome approach to determine dominant transcriptional patterns along root ontogeny zones. We found that the prevalent patterns are expressed in two opposing gradients. One is characterized by genes associated with development, the other enriched in differentiation genes. We confirmed these transcript gradients, and demonstrate that these translate to gradients in protein accumulation and gradual changes in cellular properties. We also show that gradients are genetically controlled through multiple pathways. Based on these findings, we propose that cells in the Arabidopsis root meristem gradually transition from stem cell activity toward differentiation.

Predicting gene regulatory networks by combining spatial and temporal gene expression data in Arabidopsis root stem cells.

Identifying the transcription factors (TFs) and associated networks involved in stem cell regulation is essential for understanding the initiation and growth of plant tissues and organs. Although many TFs have been shown to have a role in the Arabidopsis root stem cells, a comprehensive view of the transcriptional signature of the stem cells is lacking. In this work, we used spatial and temporal transcriptomic data to predict interactions among the genes involved in stem cell regulation. To accomplish this, we transcriptionally profiled several stem cell populations and developed a gene regulatory network inference algorithm that combines clustering with dynamic Bayesian network inference. We leveraged the topology of our networks to infer potential major regulators. Specifically, through mathematical modeling and experimental validation, we identified PERIANTHIA (PAN) as an important molecular regulator of quiescent center function. The results presented in this work show that our combination of molecular biology, computational biology, and mathematical modeling is an efficient approach to identify candidate factors that function in the stem cells.

Auxin response cell-autonomously controls ground tissue initiation in the early Arabidopsis embryo.

Plant organs are typically organized into three main tissue layers. The middle ground tissue layer comprises the majority of the plant body and serves a wide range of functions, including photosynthesis, selective nutrient uptake and storage, and gravity sensing. Ground tissue patterning and maintenance in Arabidopsis are controlled by a well-established gene network revolving around the key regulator SHORT-ROOT (SHR). In contrast, it is completely unknown how ground tissue identity is first specified from totipotent precursor cells in the embryo. The plant signaling molecule auxin, acting through AUXIN RESPONSE FACTOR (ARF) transcription factors, is critical for embryo patterning. The auxin effector ARF5/MONOPTEROS (MP) acts both cell-autonomously and noncell-autonomously to control embryonic vascular tissue formation and root initiation, respectively. Here we show that auxin response and ARF activity cell-autonomously control the asymmetric division of the first ground tissue cells. By identifying embryonic target genes, we show that MP transcriptionally initiates the ground tissue lineage and acts upstream of the regulatory network that controls ground tissue patterning and maintenance. Strikingly, whereas the SHR network depends on MP, this MP function is, at least in part, SHR independent. Our study therefore identifies auxin response as a regulator of ground tissue specification in the embryonic root, and reveals that ground tissue initiation and maintenance use different regulators and mechanisms. Moreover, our data provide a framework for the simultaneous formation of multiple cell types by the same transcriptional regulator.

Dynamic control of lateral root positioning.

In dicot root systems, lateral roots are in general regularly spaced along the longitudinal axis of the primary root to facilitate water and nutrient uptake. Recently, recurrent programmed cell death in the root cap of the growing root has been implicated in lateral root spacing. The root cap contains an auxin source that modulates lateral root patterning. Periodic release of auxin by dying root cap cells seems to trigger lateral root specification at regular intervals. However, it is currently unclear through which molecular mechanisms auxin restricts lateral root specification to specific cells along the longitudinal and radial axes of the root, or how environmental signals impact this process.

In Vivo Identification of Plant Protein Complexes Using IP-MS/MS.

Individual proteins often function as part of a protein complex. The identification of interacting proteins is therefore vital to understand the biological role and function of the studied protein. Here we describe a method for the in vivo identification of nuclear, cytoplasmic, and membrane-associated protein complexes from plant tissues using a strategy of immunoprecipitation followed by tandem mass spectrometry. By performing quantitative mass spectrometry measurements on biological triplicates, relative abundance of proteins in GFP-tagged complexes compared to background controls can be statistically evaluated to identify high-confidence interactors. We detail the entire workflow of this approach.

Cyclic programmed cell death stimulates hormone signaling and root development in Arabidopsis.

The plant root cap, surrounding the very tip of the growing root, perceives and transmits environmental signals to the inner root tissues. In Arabidopsis thaliana, auxin released by the root cap contributes to the regular spacing of lateral organs along the primary root axis. Here, we show that the periodicity of lateral organ induction is driven by recurrent programmed cell death at the most distal edge of the root cap. We suggest that synchronous bursts of cell death in lateral root cap cells release pulses of auxin to surrounding root tissues, establishing the pattern for lateral root formation. The dynamics of root cap turnover may therefore coordinate primary root growth with root branching in order to optimize the uptake of water and nutrients from the soil.

A set of domain-specific markers in the Arabidopsis embryo.

KEY MESSAGE: We describe a novel set of domain-specific markers that can be used in genetic studies, and we used two examples to show loss of stem cells in a monopteros background. Multicellular organisms can be defined by their ability to establish distinct cell identities, and it is therefore of critical importance to distinguish cell types. One step that leads to cell identity specification is activation of unique sets of transcripts. This property is often exploited in order to infer cell identity; the availability of good domain-specific marker lines is, however, poor in the Arabidopsis embryo. Here we describe a novel set of domain-specific marker lines that can be used in Arabidopsis (embryo) research. Based on transcriptomic data, we selected 12 genes for expression analysis, and according to the observed expression domain during embryogenesis, we divided them into four categories (1-ground tissue; 2-root stem cell; 3-shoot apical meristem; 4-post-embryonic). We additionally show the use of two markers from the "stem cell" category in a genetic study, where we use the absence of the markers to infer developmental defects in the monopteros mutant background. Finally, in order to judge whether the established marker lines also play a role in normal development, we generated loss-of-function resources. None of the analyzed T-DNA insertion, artificial microRNA, or misexpression lines showed any apparent phenotypic difference from wild type, indicating that these genes are not nonredundantly required for development, but also suggesting that marker activation can be considered an output of the patterning process. This set of domain-specific marker lines is therefore a valuable addition to the currently available markers and will help to move toward a generic set of tissue identity markers.

Root Cap-Derived Auxin Pre-patterns the Longitudinal Axis of the Arabidopsis Root.

During the exploration of the soil by plant roots, uptake of water and nutrients can be greatly fostered by a regular spacing of lateral roots (LRs). In the Arabidopsis root, a regular branching pattern depends on oscillatory gene activity to create prebranch sites, patches of cells competent to form LRs. Thus far, the molecular components regulating the oscillations still remain unclear. Here, we show that a local auxin source in the root cap, derived from the auxin precursor indole-3-butyric acid (IBA), modulates the oscillation amplitude, which in turn determines whether a prebranch site is created or not. Moreover, transcriptome profiling identified novel and IBA-regulated components of root patterning, such as the MEMBRANE-ASSOCIATED KINASE REGULATOR4 (MAKR4) that converts the prebranch sites into a regular spacing of lateral organs. Thus, the spatiotemporal patterning of roots is fine-tuned by the root cap-specific conversion pathway of IBA to auxin and the subsequent induction of MAKR4.