Cella: 3D data visualization for plant single-cell transcriptomics in Blender.

AIMS: Recent advancements in single-cell transcriptomics have facilitated the possibility of acquiring vast amounts of data at single-cell resolution. This development has provided a broader and more comprehensive understanding of complex biological processes. The growing datasets require a visualization tool that transforms complex data into an intuitive representation. To address this challenge, we have utilized an open-source 3D software Blender to design Cella, a cell atlas visualization tool, which transforms data into 3D heatmaps that can be rendered into image libraries. Our tool is designed to support especially research on plant development. DATA RESOURCES GENERATED: To validate our method, we have created a 3D model representing the Arabidopsis thaliana root meristem and mapped an existing single-cell RNA-seq dataset into the 3D model. This provided a user-friendly visual representation of the expression profiles of 21,489 genes from two perspectives (42,978 images). UTILITY OF THE RESOURCE: This approach is not limited to single-cell RNA-seq data of the Arabidopsis root meristem. We provide detailed step-by-step instructions to generate 3D models and a script that can be customized to project data onto different tissues. KEY RESULTS: Our tool provides a proof-of-concept method for how increasingly complex single-cell RNA-seq datasets can be visualized in a simple and cohesive manner.

Tree architecture: A strigolactone-deficient mutant reveals a connection between branching order and auxin gradient along the tree stem.

Due to their long lifespan, trees and bushes develop higher order of branches in a perennial manner. In contrast to a tall tree, with a clearly defined main stem and branching order, a bush is shorter and has a less apparent main stem and branching pattern. To address the developmental basis of these two forms, we studied several naturally occurring architectural variants in silver birch (Betula pendula). Using a candidate gene approach, we identified a bushy kanttarelli variant with a loss-of-function mutation in the BpMAX1 gene required for strigolactone (SL) biosynthesis. While kanttarelli is shorter than the wild type (WT), it has the same number of primary branches, whereas the number of secondary branches is increased, contributing to its bush-like phenotype. To confirm that the identified mutation was responsible for the phenotype, we phenocopied kanttarelli in transgenic BpMAX1::RNAi birch lines. SL profiling confirmed that both kanttarelli and the transgenic lines produced very limited amounts of SL. Interestingly, the auxin (IAA) distribution along the main stem differed between WT and BpMAX1::RNAi. In the WT, the auxin concentration formed a gradient, being higher in the uppermost internodes and decreasing toward the basal part of the stem, whereas in the transgenic line, this gradient was not observed. Through modeling, we showed that the different IAA distribution patterns may result from the difference in the number of higher-order branches and plant height. Future studies will determine whether the IAA gradient itself regulates aspects of plant architecture.

Ectopic callose deposition into woody biomass modulates the nano-architecture of macrofibrils.

Plant biomass plays an increasingly important role in the circular bioeconomy, replacing non-renewable fossil resources. Genetic engineering of this lignocellulosic biomass could benefit biorefinery transformation chains by lowering economic and technological barriers to industrial processing. However, previous efforts have mostly targeted the major constituents of woody biomass: cellulose, hemicellulose and lignin. Here we report the engineering of wood structure through the introduction of callose, a polysaccharide novel to most secondary cell walls. Our multiscale analysis of genetically engineered poplar trees shows that callose deposition modulates cell wall porosity, water and lignin contents and increases the lignin-cellulose distance, ultimately resulting in substantially decreased biomass recalcitrance. We provide a model of the wood cell wall nano-architecture engineered to accommodate the hydrated callose inclusions. Ectopic polymer introduction into biomass manifests in new physico-chemical properties and offers new avenues when considering lignocellulose engineering.

From procambium patterning to cambium activation and maintenance in the Arabidopsis root.

In addition to primary growth, which elongates the plant body, many plant species also undergo secondary growth to thicken their body. During primary vascular development, a subset of the vascular cells, called procambium and pericycle, remain undifferentiated to later gain vascular cambium and cork cambium identity, respectively. These two cambia are the lateral meristems providing secondary growth. The vascular cambium produces secondary xylem and phloem, which give plants mechanical support and transport capacity. Cork cambium produces a protective layer called cork. In this review, we focus on recent advances in understanding the formation of procambium and its gradual maturation to active cambium in the Arabidopsis thaliana root.

Gibberellins promote polar auxin transport to regulate stem cell fate decisions in cambium.

Vascular cambium contains bifacial stem cells, which produce secondary xylem to one side and secondary phloem to the other. However, how these fate decisions are regulated is unknown. Here we show that the positioning of an auxin signalling maximum within the cambium determines the fate of stem cell daughters. The position is modulated by gibberellin-regulated, PIN1-dependent polar auxin transport. Gibberellin treatment broadens auxin maximum from the xylem side of the cambium towards the phloem. As a result, xylem-side stem cell daughter preferentially differentiates into xylem, while phloem-side daughter retains stem cell identity. Occasionally, this broadening leads to direct specification of both daughters as xylem, and consequently, adjacent phloem-identity cell reverts to being stem cell. Conversely, reduced gibberellin levels favour specification of phloem-side stem cell daughter as phloem. Together, our data provide a mechanism by which gibberellin regulates the ratio of xylem and phloem production.

Epidermal injury-induced derepression of key regulator ATML1 in newly exposed cells elicits epidermis regeneration.

Plant cell fate determination depends on the relative positions of the cells in developing organisms. The shoot epidermis, the outermost cell layer of the above-ground organs in land plants, protects plants from environmental stresses. How the shoot epidermis is formed only from the outermost cells has remained unknown. Here we show that when inner leaf mesophyll cells are exposed to the surface, these cells show up-regulation of ATML1, a master regulator for epidermal cell identity in Arabidopsis thaliana. Epidermal cell types such as stomatal guard cells regenerate from young inner-lineage tissues that have a potential to accumulate ATML1 protein after epidermal injury. Surgical analyses indicate that application of pressure to the exposed site was sufficient to inhibit ATML1 derepression in the outermost mesophyll cells, suggesting this process requires pressure release. Furthermore, pharmacological analyses suggest that ATML1 derepression in the outermost mesophyll cells require cortical microtubule formation, MAPK signaling and proteasome activity. Our results suggest that surface-positional cues involving mechanical signaling are used to restrict ATML1 activity to the outermost cells and facilitate epidermal differentiation.

Mechanical conflict caused by a cell-wall-loosening enzyme activates de novo shoot regeneration.

Cellular heterogeneity is a hallmark of multicellular organisms. During shoot regeneration from undifferentiated callus, only a select few cells, called progenitors, develop into shoot. How these cells are selected and what governs their subsequent progression to a patterned organ system is unknown. Using Arabidopsis thaliana, we show that it is not just the abundance of stem cell regulators but rather the localization pattern of polarity proteins that predicts the progenitor's fate. A shoot-promoting factor, CUC2, activated the expression of the cell-wall-loosening enzyme, XTH9, solely in a shell of cells surrounding the progenitor, causing different mechanical stresses in these cells. This mechanical conflict then activates cell polarity in progenitors to promote meristem formation. Interestingly, genetic or physical perturbations to cells surrounding the progenitor impaired the progenitor and vice versa. These suggest a feedback loop between progenitors and their neighbors for shoot regeneration in the absence of tissue-patterning cues.

Analysis of exocyst function in endodermis reveals its widespread contribution and specificity of action.

The exocyst is the main plasma membrane vesicle-tethering complex in eukaryotes and is composed of eight different subunits. Yet, in plant genomes, many subunits display multiple copies, thought to reflect evolution of complex subtypes with divergent functions. In Arabidopsis thaliana root endodermal cells, the isoform EXO70A1 is required for positioning of CASP1 at the Casparian Strip Domain, but not for its non-targeted secretion to the plasma membrane. Here, we show that exo84b resembles exo70a1 mutants regarding CASP1 mistargeting and secretion of apoplastic proteins, but exo84b additionally affects secretion of other integral plasma membrane proteins. Moreover, conditional, cell-type-specific gene editing of the single-copy core component SEC6 allows visualization of secretion defects in plant cells with a complete lack of exocyst complex function. Our approach opens avenues for deciphering the complexity/diversity of exocyst functions in plant cells and enables analysis of central trafficking components with lethal phenotypes.

The Making of Plant Armor: The Periderm.

The periderm acts as armor protecting the plant's inner tissues from biotic and abiotic stress. It forms during the radial thickening of plant organs such as stems and roots and replaces the function of primary protective tissues such as the epidermis and the endodermis. A wound periderm also forms to heal and protect injured tissues. The periderm comprises a meristematic tissue called the phellogen, or cork cambium, and its derivatives: the lignosuberized phellem and the phelloderm. Research on the periderm has mainly focused on the chemical composition of the phellem due to its relevance as a raw material for industrial processes. Today, there is increasing interest in the regulatory network underlying periderm development as a novel breeding trait to improve plant resilience and to sequester CO2. Here, we discuss our current understanding of periderm formation, focusing on aspects of periderm evolution, mechanisms of periderm ontogenesis, regulatory networks underlying phellogen initiation and cork differentiation, and future challenges of periderm research.

Means to Quantify Vascular Cell File Numbers in Different Tissues.

Oriented cell divisions are crucial throughout plant development to define the final size and shape of organs and tissues. As most of the tissues in mature roots and stems are derived from vascular tissues, studying cell proliferation in the vascular cell lineage is of great importance. Although perturbations of vascular development are often visible already at the whole plant macroscopic phenotype level, a more detailed characterization of the vascular anatomy, cellular organization, and differentiation status of specific vascular cell types can provide insights into which pathway or developmental program is affected. In particular, defects in the frequency or orientation of cell divisions can be reliably identified from the number of vascular cell files. Here, we provide a detailed description of the different clearing, staining, and imaging techniques that allow precise phenotypic analysis of vascular tissues in different organs of the model plant Arabidopsis thaliana throughout development, including the quantification of cell file numbers, differentiation status of vascular cell types, and expression of reporter genes.

Cell-by-cell dissection of phloem development links a maturation gradient to cell specialization.

In the plant meristem, tissue-wide maturation gradients are coordinated with specialized cell networks to establish various developmental phases required for indeterminate growth. Here, we used single-cell transcriptomics to reconstruct the protophloem developmental trajectory from the birth of cell progenitors to terminal differentiation in the Arabidopsis thaliana root. PHLOEM EARLY DNA-BINDING-WITH-ONE-FINGER (PEAR) transcription factors mediate lineage bifurcation by activating guanosine triphosphatase signaling and prime a transcriptional differentiation program. This program is initially repressed by a meristem-wide gradient of PLETHORA transcription factors. Only the dissipation of PLETHORA gradient permits activation of the differentiation program that involves mutual inhibition of early versus late meristem regulators. Thus, for phloem development, broad maturation gradients interface with cell-type-specific transcriptional regulators to stage cellular differentiation.

Cytokinins initiate secondary growth in the Arabidopsis root through a set of LBD genes.

During primary growth, plant tissues increase their length, and as these tissues mature, they initiate secondary growth to increase thickness.1 It is not known what activates this transition to secondary growth. Cytokinins are key plant hormones regulating vascular development during both primary and secondary growth. During primary growth of Arabidopsis roots, cytokinins promote procambial cell proliferation2,3 and vascular patterning together with the hormone auxin.4-7 In the absence of cytokinins, secondary growth fails to initiate.8 Enhanced cytokinin levels, in turn, promote secondary growth.8,9 Despite the importance of cytokinins, little is known about the downstream signaling events in this process. Here, we show that cytokinins and a few downstream LATERAL ORGAN BOUNDARIES DOMAIN (LBD) family of transcription factors are rate-limiting components in activating and further promoting secondary growth in Arabidopsis roots. Cytokinins directly activate transcription of two homologous LBD genes, LBD3 and LBD4. Two other homologous LBDs, LBD1 and LBD11, are induced only after prolonged cytokinin treatment. Our genetic studies revealed a two-stage mechanism downstream of cytokinin signaling: while LBD3 and LBD4 regulate activation of secondary growth, LBD1, LBD3, LBD4, and LBD11 together promote further radial growth and maintenance of cambial stem cells. LBD overexpression promoted rapid cell growth followed by accelerated cell divisions, thus leading to enhanced secondary growth. Finally, we show that LBDs rapidly inhibit cytokinin signaling. Together, our data suggest that the cambium-promoting LBDs negatively feed back into cytokinin signaling to keep root secondary growth in balance.

What is quantitative plant biology?

Quantitative plant biology is an interdisciplinary field that builds on a long history of biomathematics and biophysics. Today, thanks to high spatiotemporal resolution tools and computational modelling, it sets a new standard in plant science. Acquired data, whether molecular, geometric or mechanical, are quantified, statistically assessed and integrated at multiple scales and across fields. They feed testable predictions that, in turn, guide further experimental tests. Quantitative features such as variability, noise, robustness, delays or feedback loops are included to account for the inner dynamics of plants and their interactions with the environment. Here, we present the main features of this ongoing revolution, through new questions around signalling networks, tissue topology, shape plasticity, biomechanics, bioenergetics, ecology and engineering. In the end, quantitative plant biology allows us to question and better understand our interactions with plants. In turn, this field opens the door to transdisciplinary projects with the society, notably through citizen science.

A network of transcriptional repressors modulates auxin responses.

The regulation of signalling capacity, combined with the spatiotemporal distribution of developmental signals themselves, is pivotal in setting developmental responses in both plants and animals1. The hormone auxin is a key signal for plant growth and development that acts through the AUXIN RESPONSE FACTOR (ARF) transcription factors2-4. A subset of these, the conserved class A ARFs5, are transcriptional activators of auxin-responsive target genes that are essential for regulating auxin signalling throughout the plant lifecycle2,3. Although class A ARFs have tissue-specific expression patterns, how their expression is regulated is unknown. Here we show, by investigating chromatin modifications and accessibility, that loci encoding these proteins are constitutively open for transcription. Through yeast one-hybrid screening, we identify the transcriptional regulators of the genes encoding class A ARFs from Arabidopsis thaliana and demonstrate that each gene is controlled by specific sets of transcriptional regulators. Transient transformation assays and expression analyses in mutants reveal that, in planta, the majority of these regulators repress the transcription of genes encoding class A ARFs. These observations support a scenario in which the default configuration of open chromatin enables a network of transcriptional repressors to regulate expression levels of class A ARF proteins and modulate auxin signalling output throughout development.

Plant Biology: Storage Root Growth through Thick and Thin.

Embedded within plant stems and roots, the radially dividing cambium controls organ thickening. A study of radish root transcriptomes finds that multiple cambial gene regulatory networks are conserved between radish and the model species Arabidopsis. These results highlight the close relationship between root thickening and environmental responses.

An inducible genome editing system for plants.

Conditional manipulation of gene expression is a key approach to investigating the primary function of a gene in a biological process. While conditional and cell-type-specific overexpression systems exist for plants, there are currently no systems available to disable a gene completely and conditionally. Here, we present a new tool with which target genes can efficiently and conditionally be knocked out by genome editing at any developmental stage. Target genes can also be knocked out in a cell-type-specific manner. Our tool is easy to construct and will be particularly useful for studying genes having null alleles that are non-viable or show pleiotropic developmental defects.

A coherent feed-forward loop drives vascular regeneration in damaged aerial organs of plants growing in a normal developmental context.

Aerial organs of plants, being highly prone to local injuries, require tissue restoration to ensure their survival. However, knowledge of the underlying mechanism is sparse. In this study, we mimicked natural injuries in growing leaves and stems to study the reunion between mechanically disconnected tissues. We show that PLETHORA (PLT) and AINTEGUMENTA (ANT) genes, which encode stem cell-promoting factors, are activated and contribute to vascular regeneration in response to these injuries. PLT proteins bind to and activate the CUC2 promoter. PLT proteins and CUC2 regulate the transcription of the local auxin biosynthesis gene YUC4 in a coherent feed-forward loop, and this process is necessary to drive vascular regeneration. In the absence of this PLT-mediated regeneration response, leaf ground tissue cells can neither acquire the early vascular identity marker ATHB8, nor properly polarise auxin transporters to specify new venation paths. The PLT-CUC2 module is required for vascular regeneration, but is dispensable for midvein formation in leaves. We reveal the mechanisms of vascular regeneration in plants and distinguish between the wound-repair ability of the tissue and its formation during normal development.