Localizing Total mRNA in Plant Cells.

Visualizing the location of the total cellular mRNA pool can be important to understand how different genes affect cellular physiology. Over the past decade researchers investigating RNA processing, nuclear transport and the function of the nuclear pore complex have used in situ hybridization protocol to visualize and quantify the accumulation of the total mRNA pool within the plant cell nucleus.

The INDEPTH (Impact of Nuclear Domains on Gene Expression and Plant Traits) Academy: a community resource for plant science.

This Community Resource paper introduces the range of materials developed by the INDEPTH (Impact of Nuclear Domains on Gene Expression and Plant Traits) COST Action made available through the INDEPTH Academy. Recent rapid growth in understanding of the significance of epigenetic controls in plant and crop science has led to a need for shared, high-quality resources, standardization of protocols, and repositories for open access data. The INDEPTH Academy provides a range of masterclass tutorials, standardized protocols, and teaching webinars, together with a rapidly developing repository to support imaging and spatial analysis of the nucleus and deep learning for automated analysis. These resources were developed partly as a response to the COVID-19 pandemic, but also driven by needs and opportunities identified by the INDEPTH community of ~200 researchers in 80 laboratories from 32 countries. This community report outlines the resources produced and how they will be extended beyond the INDEPTH project, but also aims to encourage the wider community to engage with epigenetics and nuclear structure by accessing these resources.

Anno genominis XX: 20 years of Arabidopsis genomics.

Twenty years ago, the Arabidopsis thaliana genome sequence was published. This was an important moment as it was the first sequenced plant genome and explicitly brought plant science into the genomics era. At the time, this was not only an outstanding technological achievement, but it was characterized by a superb global collaboration. The Arabidopsis genome was the seed for plant genomic research. Here, we review the development of numerous resources based on the genome that have enabled discoveries across plant species, which has enhanced our understanding of how plants function and interact with their environments.

How to build an effective research network: lessons from two decades of the GARNet plant science community.

Successful collaborative research is dependent on excellent ideas and innovative experimental approaches, as well as the provision of appropriate support networks. Collaboration requires venues, infrastructures, training facilities, and, perhaps most importantly, a sustained commitment to work together as a community. These activities do not occur without significant effort, yet can be facilitated and overseen by the leadership of a research network that has a clearly defined role to help build resources for their community. Over the past 20 years, this is a role that the UKRI-BBSRC-funded GARNet network has played in the support of the UK curiosity-driven, discovery-led plant science research community. This article reviews the lessons learnt by GARNet in the hope that they can inform the practical implementation of current and future research networks.

Current status of the multinational Arabidopsis community.

The multinational Arabidopsis research community is highly collaborative and over the past thirty years these activities have been documented by the Multinational Arabidopsis Steering Committee (MASC). Here, we (a) highlight recent research advances made with the reference plant Arabidopsis thaliana; (b) provide summaries from recent reports submitted by MASC subcommittees, projects and resources associated with MASC and from MASC country representatives; and (c) initiate a call for ideas and foci for the "fourth decadal roadmap," which will advise and coordinate the global activities of the Arabidopsis research community.

Low Phosphate Puts Auxin in the Root Hairs.

The molecular changes that allow plant roots to response to low phosphate levels are poorly understood. A series of three papers investigate this phenomenon and reveal which components of the auxin response are key for transmitting the phosphate signal into changes in root hair phenotypes.

Meeting report - INDEPTH kick-off meeting.

The precise location of chromatin domains within the cell nucleus has seen growing recognition in the past decade as an additional mechanism of controlling gene expression in both plants and animals (Dekker et al., 2017). Consequently, international efforts are devoted to understanding the organising principle of this organelle in plants, and notably the nature and the role of functional compartments on gene expression (Graumann et al., 2013; Sotelo-Silveira et al., 2018). The European cooperation 'Impact of Nuclear Domains on Gene Expression and Plant Traits' (INDEPTH) brings together molecular cell biologists, plant physiologists, bioinformaticians, image analysts and computer scientists. They aim to address the question of how nuclear architecture, chromatin organisation and gene expression are connected in plants, particularly in relation to traits of interest such as biomass, reproduction and resistance to pathogens (https://www.brookes.ac.uk/indepth/). The kick-off meeting of the INDEPTH consortium took place in Clermont-Ferrand, France, on 12-14th March 2018, where more than 80 researchers set the agenda for the coming four years of research and collaboration.

Meeting report: GARNet/OpenPlant CRISPR-Cas workshop.

Targeted genome engineering has been described as a "game-changing technology" for fields as diverse as human genetics and plant biotechnology. One technique used for precise gene editing utilises the CRISPR-Cas system and is an effective method for genetic engineering in a wide variety of plants. However, many researchers remain unaware of both the technical challenges that emerge when using this technique or of its potential benefits. Therefore in September 2015, GARNet and OpenPlant organized a two-day workshop at the John Innes Centre that provided both background information and hands-on training for this important technology.

Overexpressing components of the nuclear transport apparatus causes severe growth symptoms in tobacco leaves.

Regulating nucleo-cytoplasmic transport of RNA and protein is a key cellular control point. Perturbing the function of plant nuclear transport components can cause significant developmental defects and in this report we add an important line to this evidence. Overexpression of AtRAN1 or AtNUP62 in Nicotiana benthamiana causes significant damage to leaf tissue. This demonstrates that the precise control of nuclear transport is an important aspect of maintaining tissue integrity.

The plant nuclear envelope and regulation of gene expression.

The nuclear envelope (NE) separates the key mechanisms of transcription and translation, and as such is a critical control point in all eukaryotic cells. In plants, the proteins of the NE influence a number of processes including the control of nucleo-cytoplasmic transport of RNA and protein, chromatin localization to the nuclear periphery, and direct chromatin binding by members of the nuclear pore complex (NPC). In this review I attempt to bring these roles under the umbrella of their effect on gene expression, even though the complex nature of this cellular environment means there is considerable overlap of effects. Although the volume of research in plant cells has greatly improved over recent years, it is clear that our understanding of how the components of the NE either directly or indirectly influence gene expression is still in its infancy.

Components of the Arabidopsis nuclear pore complex play multiple diverse roles in control of plant growth.

The nuclear pore complex (NPC) is a multisubunit protein conglomerate that facilitates movement of RNA and protein between the nucleus and cytoplasm. Relatively little is known regarding the influence of the Arabidopsis NPC on growth and development. Seedling development, flowering time, nuclear morphology, mRNA accumulation, and gene expression changes in Arabidopsis nucleoporin mutants were investigated. Nuclear export of mRNA is differentially affected in plants with defects in nucleoporins that lie in different NPC subcomplexes. This study reveals differences in the manner by which nucleoporins alter molecular and plant growth phenotypes, suggesting that nuclear pore subcomplexes play distinct roles in nuclear transport and reveal a possible feedback relationship between the expression of genes involved in nuclear transport.

SUNrises on the International Plant Nucleus Consortium: SEB Salzburg 2012.

The nuclear periphery is a dynamic, structured environment, whose precise functions are essential for global processes-from nuclear, to cellular, to organismal. Its main components-the nuclear envelope (NE) with inner and outer nuclear membranes (INM and ONM), nuclear pore complexes (NPC), associated cytoskeletal and nucleoskeletal components as well as chromatin are conserved across eukaryotes (Fig. 1). In metazoans in particular, the structure and functions of nuclear periphery components are intensely researched partly because of their involvement in various human diseases. While far less is known about these in plants, the last few years have seen a significant increase in research activity in this area. Plant biologists are not only catching up with the animal field, but recent findings are pushing our advances in this field globally. In recognition of this developing field, the Annual Society of Experimental Biology Meeting in Salzburg kindly hosted a session co-organized by Katja Graumann and David E. Evans (Oxford Brookes University) highlighting new insights into plant nuclear envelope proteins and their interactions. This session brought together leading researchers with expertise in topics such as epigenetics, meiosis, nuclear pore structure and functions, nucleoskeleton and nuclear envelope composition. An open and friendly exchange of ideas was fundamental to the success of the meeting, which resulted in founding the International Plant Nucleus Consortium. This review highlights new developments in plant nuclear envelope research presented at the conference and their importance for the wider understanding of metazoan, yeast and plant nuclear envelope functions and properties.

Assessing the function of the plant nuclear pore complex and the search for specificity.

Plant cells encounter a wide variety of molecules that influence their gene expression and development. A key component of most signal transduction pathways involves the regulated movement of molecules into and out of the nucleus. The plant nuclear pore complex (NPC) is a critical controlling element in this nucleocytoplasmic movement of protein and RNA. The NPC is comprised of approximately 30 nucleoporin proteins arranged in radial symmetry around the central pore. Over recent years our understanding of how the NPC impacts different signalling pathways has increased following the identification of a range of nucleoporin mutant plants. These mutants allow us to gain insight into how the response to hormonal, abiotic, and biotic stresses are effected by changes in nuclear transport. Importantly we have little information regarding the specific molecules whose nuclear transport is altered in these processes and the identification of these proteins is a significant challenge. Here is presented an overview as to how the members of the plant NPC affect signalling pathways, highlighting the progress and difficulties within this research area.

A combinatorial TIR1/AFB-Aux/IAA co-receptor system for differential sensing of auxin.

The plant hormone auxin regulates virtually every aspect of plant growth and development. Auxin acts by binding the F-box protein transport inhibitor response 1 (TIR1) and promotes the degradation of the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) transcriptional repressors. Here we show that efficient auxin binding requires assembly of an auxin co-receptor complex consisting of TIR1 and an Aux/IAA protein. Heterologous experiments in yeast and quantitative IAA binding assays using purified proteins showed that different combinations of TIR1 and Aux/IAA proteins form co-receptor complexes with a wide range of auxin-binding affinities. Auxin affinity seems to be largely determined by the Aux/IAA. As there are 6 TIR1/AUXIN SIGNALING F-BOX proteins (AFBs) and 29 Aux/IAA proteins in Arabidopsis thaliana, combinatorial interactions may result in many co-receptors with distinct auxin-sensing properties. We also demonstrate that the AFB5-Aux/IAA co-receptor selectively binds the auxinic herbicide picloram. This co-receptor system broadens the effective concentration range of the hormone and may contribute to the complexity of auxin response.

Root gravitropism is regulated by a transient lateral auxin gradient controlled by a tipping-point mechanism.

Gravity profoundly influences plant growth and development. Plants respond to changes in orientation by using gravitropic responses to modify their growth. Cholodny and Went hypothesized over 80 years ago that plants bend in response to a gravity stimulus by generating a lateral gradient of a growth regulator at an organ's apex, later found to be auxin. Auxin regulates root growth by targeting Aux/IAA repressor proteins for degradation. We used an Aux/IAA-based reporter, domain II (DII)-VENUS, in conjunction with a mathematical model to quantify auxin redistribution following a gravity stimulus. Our multidisciplinary approach revealed that auxin is rapidly redistributed to the lower side of the root within minutes of a 90° gravity stimulus. Unexpectedly, auxin asymmetry was rapidly lost as bending root tips reached an angle of 40° to the horizontal. We hypothesize roots use a "tipping point" mechanism that operates to reverse the asymmetric auxin flow at the midpoint of root bending. These mechanistic insights illustrate the scientific value of developing quantitative reporters such as DII-VENUS in conjunction with parameterized mathematical models to provide high-resolution kinetics of hormone redistribution.

The auxin signalling network translates dynamic input into robust patterning at the shoot apex.

The plant hormone auxin is thought to provide positional information for patterning during development. It is still unclear, however, precisely how auxin is distributed across tissues and how the hormone is sensed in space and time. The control of gene expression in response to auxin involves a complex network of over 50 potentially interacting transcriptional activators and repressors, the auxin response factors (ARFs) and Aux/IAAs. Here, we perform a large-scale analysis of the Aux/IAA-ARF pathway in the shoot apex of Arabidopsis, where dynamic auxin-based patterning controls organogenesis. A comprehensive expression map and full interactome uncovered an unexpectedly simple distribution and structure of this pathway in the shoot apex. A mathematical model of the Aux/IAA-ARF network predicted a strong buffering capacity along with spatial differences in auxin sensitivity. We then tested and confirmed these predictions using a novel auxin signalling sensor that reports input into the signalling pathway, in conjunction with the published DR5 transcriptional output reporter. Our results provide evidence that the auxin signalling network is essential to create robust patterns at the shoot apex.