In-Depth Quantification of Cell Division and Elongation Dynamics at the Tip of Growing Arabidopsis Roots Using 4D Microscopy, AI-Assisted Image Processing and Data Sonification.

One of the fundamental questions in plant developmental biology is how cell proliferation and cell expansion coordinately determine organ growth and morphology. An amenable system to address this question is the Arabidopsis root tip, where cell proliferation and elongation occur in spatially separated domains, and cell morphologies can easily be observed using a confocal microscope. While past studies revealed numerous elements of root growth regulation including gene regulatory networks, hormone transport and signaling, cell mechanics and environmental perception, how cells divide and elongate under possible constraints from cell lineages and neighboring cell files has not been analyzed quantitatively. This is mainly due to the technical difficulties in capturing cell division and elongation dynamics at the tip of growing roots, as well as an extremely labor-intensive task of tracing the lineages of frequently dividing cells. Here, we developed a motion-tracking confocal microscope and an Artificial Intelligence (AI)-assisted image-processing pipeline that enables semi-automated quantification of cell division and elongation dynamics at the tip of vertically growing Arabidopsis roots. We also implemented a data sonification tool that facilitates human recognition of cell division synchrony. Using these tools, we revealed previously unnoted lineage-constrained dynamics of cell division and elongation, and their contribution to the root zonation boundaries.

Patterned proliferation orients tissue-wide stress to control root vascular symmetry in Arabidopsis.

Symmetric tissue alignment is pivotal to the functions of plant vascular tissue, such as long-distance molecular transport and lateral organ formation. During the vascular development of the Arabidopsis roots, cytokinins initially determine cell-type boundaries among vascular stem cells and subsequently promote cell proliferation to establish vascular tissue symmetry. Although it is unknown whether and how the symmetry of initially defined boundaries is progressively refined under tissue growth in plants, such boundary shapes in animal tissues are regulated by cell fluidity, e.g., cell migration and intercalation, lacking in plant tissues. Here, we uncover that cell proliferation during vascular development produces anisotropic compressive stress, smoothing, and symmetrizing cell arrangement of the vascular-cell-type boundary. Mechanistically, the GATA transcription factor HANABA-TARANU cooperates with the type-B Arabidopsis response regulators to form an incoherent feedforward loop in cytokinin signaling. The incoherent feedforward loop fine-tunes the position and frequency of vascular cell proliferation, which in turn restricts the source of mechanical stress to the position distal and symmetric to the boundary. By combinatorial analyses of mechanical simulations and laser cell ablation, we show that the spatially constrained environment of vascular tissue efficiently entrains the stress orientation among the cells to produce a tissue-wide stress field. Together, our data indicate that the localized proliferation regulated by the cytokinin signaling circuit is decoded into a globally oriented mechanical stress to shape the vascular tissue symmetry, representing a reasonable mechanism controlling the boundary alignment and symmetry in tissue lacking cell fluidity.

Autophagy promotes organelle clearance and organized cell separation of living root cap cells in Arabidopsis thaliana.

The root cap is a multilayered tissue covering the tip of a plant root that directs root growth through its unique functions, such as gravity sensing and rhizosphere interaction. To maintain the structure and function of the root cap, its constituent cells are constantly turned over through balanced cell division and cell detachment in the inner and outer cell layers, respectively. Upon displacement toward the outermost layer, columella cells at the central root cap domain functionally transition from gravity-sensing cells to secretory cells, but the mechanisms underlying this drastic cell fate transition are largely unknown. Here, using live-cell tracking microscopy, we show that organelles in the outermost cell layer undergo dramatic rearrangements. This rearrangement depends, at least partially, on spatiotemporally regulated activation of autophagy. Notably, this root cap autophagy does not lead to immediate cell death, but is instead necessary for organized separation of living root cap cells, highlighting a previously undescribed role of developmentally regulated autophagy in plants. This article has an associated 'The people behind the papers' interview.

SHORTROOT-Mediated Intercellular Signals Coordinate Phloem Development in Arabidopsis Roots.

Asymmetric cell division (ACD) and positional signals play critical roles in the tissue patterning process. In the Arabidopsis (Arabidopsis thaliana) root meristem, two major phloem cell types arise via ACDs of distinct origins: one for companion cells (CCs) and the other for proto- and metaphloem sieve elements (SEs). The molecular mechanisms underlying each of these processes have been reported; however, how these are coordinated has remained elusive. Here, we report a new phloem development process coordinated via the SHORTROOT (SHR) transcription factor in Arabidopsis. The movement of SHR into the endodermis regulates the ACD for CC formation by activating microRNA165/6, while SHR moving into the phloem regulates the ACD generating the two phloem SEs. In the phloem, SHR sequentially activates NAC-REGULATED SEED MORPHOLOGY 1 (NARS1) and SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 2 (SND2), and these three together form a positive feedforward loop. Under this regulatory scheme, NARS1, generated in the CCs of the root differentiation zone, establishes a top-down signal that drives the ACD for phloem SEs in the meristem. SND2 appears to function downstream to amplify NARS1 via positive feedback. This new regulatory mechanism expands our understanding of the sophisticated vascular tissue patterning processes occurring during postembryonic root development.plantcell;32/5/1519/FX1F1fx1.

A core mechanism for specifying root vascular patterning can replicate the anatomical variation seen in diverse plant species.

Pattern formation is typically controlled through the interaction between molecular signals within a given tissue. During early embryonic development, roots of the model plant Arabidopsis thaliana have a radially symmetric pattern, but a heterogeneous input of the hormone auxin from the two cotyledons forces the vascular cylinder to develop a diarch pattern with two xylem poles. Molecular analyses and mathematical approaches have uncovered the regulatory circuit that propagates this initial auxin signal into a stable cellular pattern. The diarch pattern seen in Arabidopsis is relatively uncommon among flowering plants, with most species having between three and eight xylem poles. Here, we have used multiscale mathematical modelling to demonstrate that this regulatory module does not require a heterogeneous auxin input to specify the vascular pattern. Instead, the pattern can emerge dynamically, with its final form dependent upon spatial constraints and growth. The predictions of our simulations compare to experimental observations of xylem pole number across a range of species, as well as in transgenic systems in Arabidopsis in which we manipulate the size of the vascular cylinder. By considering the spatial constraints, our model is able to explain much of the diversity seen in different flowering plant species.

DOF2.1 Controls Cytokinin-Dependent Vascular Cell Proliferation Downstream of TMO5/LHW.

To create a three-dimensional structure, plants rely on oriented cell divisions and cell elongation. Oriented cell divisions are specifically important in procambium cells of the root to establish the different vascular cell types [1, 2]. These divisions are in part controlled by the auxin-controlled TARGET OF MONOPTEROS5 (TMO5) and LONESOME HIGHWAY (LHW) transcription factor complex [3-7]. Loss-of-function of tmo5 or lhw clade members results in strongly reduced vascular cell file numbers, whereas ectopic expression of both TMO5 and LHW can ubiquitously induce periclinal and radial cell divisions in all cell types of the root meristem. TMO5 and LHW interact only in young xylem cells, where they promote expression of two direct target genes involved in the final step of cytokinin (CK) biosynthesis, LONELY GUY3 (LOG3) and LOG4 [8, 9] Therefore, CK was hypothesized to act as a mobile signal from the xylem to trigger divisions in the neighboring procambium cells [3, 6]. To unravel how TMO5/LHW-dependent cytokinin regulates cell proliferation, we analyzed the transcriptional responses upon simultaneous induction of both transcription factors. Using inferred network analysis, we identified AT2G28510/DOF2.1 as a cytokinin-dependent downstream target gene. We further showed that DOF2.1 controls specific procambium cell divisions without inducing other cytokinin-dependent effects such as the inhibition of vascular differentiation. In summary, our results suggest that DOF2.1 and its closest homologs control vascular cell proliferation, thus leading to radial expansion of the root.

High levels of auxin signalling define the stem-cell organizer of the vascular cambium.

Wood, a type of xylem tissue, originates from cell proliferation of the vascular cambium. Xylem is produced inside, and phloem outside, of the cambium1. Morphogenesis in plants is typically coordinated by organizer cells that direct the adjacent stem cells to undergo programmed cell division and differentiation. The location of the vascular cambium stem cells and whether the organizer concept applies to the cambium are currently unknown2. Here, using lineage-tracing and molecular genetic studies in the roots of Arabidopsis thaliana, we show that cells with a xylem identity direct adjacent vascular cambial cells to divide and function as stem cells. Thus, these xylem-identity cells constitute an organizer. A local maximum of the phytohormone auxin, and consequent expression of CLASS III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) transcription factors, promotes xylem identity and cellular quiescence of the organizer cells. Additionally, the organizer maintains phloem identity in a non-cell-autonomous fashion. Consistent with this dual function of the organizer cells, xylem and phloem originate from a single, bifacial stem cell in each radial cell file, which confirms the classical theory of a uniseriate vascular cambium3. Clones that display high levels of ectopically activated auxin signalling differentiate as xylem vessels; these clones induce cell divisions and the expression of cambial and phloem markers in the adjacent cells, which suggests that a local auxin-signalling maximum is sufficient to specify a stem-cell organizer. Although vascular cambium has a unique function among plant meristems, the stem-cell organizer of this tissue shares features with the organizers of root and shoot meristems.

Mobile PEAR transcription factors integrate positional cues to prime cambial growth.

Apical growth in plants initiates upon seed germination, whereas radial growth is primed only during early ontogenesis in procambium cells and activated later by the vascular cambium1. Although it is not known how radial growth is organized and regulated in plants, this system resembles the developmental competence observed in some animal systems, in which pre-existing patterns of developmental potential are established early on2,3. Here we show that in Arabidopsis the initiation of radial growth occurs around early protophloem-sieve-element cell files of the root procambial tissue. In this domain, cytokinin signalling promotes the expression of a pair of mobile transcription factors-PHLOEM EARLY DOF 1 (PEAR1) and PHLOEM EARLY DOF 2 (PEAR2)-and their four homologues (DOF6, TMO6, OBP2 and HCA2), which we collectively name PEAR proteins. The PEAR proteins form a short-range concentration gradient that peaks at protophloem sieve elements, and activates gene expression that promotes radial growth. The expression and function of PEAR proteins are antagonized by the HD-ZIP III proteins, well-known polarity transcription factors4-the expression of which is concentrated in the more-internal domain of radially non-dividing procambial cells by the function of auxin, and mobile miR165 and miR166 microRNAs. The PEAR proteins locally promote transcription of their inhibitory HD-ZIP III genes, and thereby establish a negative-feedback loop that forms a robust boundary that demarks the zone of cell division. Taken together, our data establish that during root procambial development there exists a network in which a module that links PEAR and HD-ZIP III transcription factors integrates spatial information of the hormonal domains and miRNA gradients to provide adjacent zones of dividing and more-quiescent cells, which forms a foundation for further radial growth.

Interactions between callose and cellulose revealed through the analysis of biopolymer mixtures.

The properties of (1,3)-ß-glucans (i.e., callose) remain largely unknown despite their importance in plant development and defence. Here we use mixtures of (1,3)-ß-glucan and cellulose, in ionic liquid solution and hydrogels, as proxies to understand the physico-mechanical properties of callose. We show that after callose addition the stiffness of cellulose hydrogels is reduced at a greater extent than predicted from the ideal mixing rule (i.e., the weighted average of the individual components' properties). In contrast, yield behaviour after the elastic limit is more ductile in cellulose-callose hydrogels compared with sudden failure in 100% cellulose hydrogels. The viscoelastic behaviour and the diffusion of the ions in mixed ionic liquid solutions strongly indicate interactions between the polymers. Fourier-transform infrared analysis suggests that these interactions impact cellulose organisation in hydrogels and cell walls. We conclude that polymer interactions alter the properties of callose-cellulose mixtures beyond what it is expected by ideal mixing.

Transcriptome dynamics at Arabidopsis graft junctions reveal an intertissue recognition mechanism that activates vascular regeneration.

The ability for cut tissues to join and form a chimeric organism is a remarkable property of many plants; however, grafting is poorly characterized at the molecular level. To better understand this process, we monitored genome-wide gene expression changes in grafted Arabidopsis thaliana hypocotyls. We observed a sequential activation of genes associated with cambium, phloem, and xylem formation. Tissues above and below the graft rapidly developed an asymmetry such that many genes were more highly expressed on one side than on the other. This asymmetry correlated with sugar-responsive genes, and we observed an accumulation of starch above the graft junction. This accumulation decreased along with asymmetry once the sugar-transporting vascular tissues reconnected. Despite the initial starvation response below the graft, many genes associated with vascular formation were rapidly activated in grafted tissues but not in cut and separated tissues, indicating that a recognition mechanism was activated independently of functional vascular connections. Auxin, which is transported cell to cell, had a rapidly elevated response that was symmetric, suggesting that auxin was perceived by the root within hours of tissue attachment to activate the vascular regeneration process. A subset of genes was expressed only in grafted tissues, indicating that wound healing proceeded via different mechanisms depending on the presence or absence of adjoining tissues. Such a recognition process could have broader relevance for tissue regeneration, intertissue communication, and tissue fusion events.

Functionally Diversified Members of the MIR165/6 Gene Family Regulate Ovule Morphogenesis in Arabidopsis thaliana.

The ovules of flowering plants consist of a central embryo sac and surrounding layers of the inner and outer integument. As these structural units eventually give rise to the embryo/endosperm and seed coat, respectively, a precisely organized ovule structure is essential for successful fertilization and seed production. In Arabidopsis thaliana, correct ovule patterning depends on the restricted expression of the CLASS III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIP III) gene PHABULOSA (PHB) in the apical region of the incipient inner integument, which in turn is regulated via post-transcriptional suppression by miR165 and miR166 (miR165/6) derived from multiple MIR165/6 genes. While a common subset of MIR165/6 genes regulate PHB expression in the root meristem, leaf primordium and embryo, it is unknown whether the same MIR165/6 subset also regulate PHB expression during ovule development. Furthermore, it is unclear where in the ovule primordia miR165/6 are produced. Here, we show that a distinct set of MIR165/6 genes that are highly expressed in the small regions of early ovule primordia restrict the PHB expression domain to promote integument formation. MIR165/6 genes that function in ovule development are phylogenetically distinct from those acting in roots and leaf primordia. Taken together, our data suggest that members of the MIR165/6 gene family are diversified in their expression capacity to establish elaborate PHB expression patterns depending on the developmental context, thereby allowing HD-ZIP III transcription factors to regulate multiple aspects of plant development.

Control of root cap maturation and cell detachment by BEARSKIN transcription factors in Arabidopsis.

The root cap supports root growth by protecting the root meristem, sensing gravity and interacting with the rhizosphere through metabolite secretion and cell dispersal. Sustained root cap functions therefore rely on balanced proliferation of proximal stem cells and regulated detachment of distal mature cells. Although the gene regulatory network that governs stem cell activity in the root cap has been extensively studied in Arabidopsis, the mechanisms by which root cap cells mature and detach from the root tip are poorly understood. We performed a detailed expression analysis of three regulators of root cap differentiation, SOMBRERO, BEARSKIN1 and BEARSKIN2, and identified their downstream genes. Our results indicate that expression of BEARSKIN1 and BEARSKIN2 is associated with cell positioning on the root surface. We identified a glycosyl hydrolase 28 (GH28) family polygalacturonase (PG) gene as a direct target of BEARSKIN1. Overexpression and loss-of-function analyses demonstrated that the protein encoded by this PG gene facilitates cell detachment. We thus revealed a molecular link between the key regulators of root cap differentiation and the cellular events underlying root cap-specific functions.

AINTEGUMENTA and the D-type cyclin CYCD3;1 regulate root secondary growth and respond to cytokinins.

Higher plant vasculature is characterized by two distinct developmental phases. Initially, a well-defined radial primary pattern is established. In eudicots, this is followed by secondary growth, which involves development of the cambium and is required for efficient water and nutrient transport and wood formation. Regulation of secondary growth involves several phytohormones, and cytokinins have been implicated as key players, particularly in the activation of cell proliferation, but the molecular mechanisms mediating this hormonal control remain unknown. Here we show that the genes encoding the transcription factor AINTEGUMENTA (ANT) and the D-type cyclin CYCD3;1 are expressed in the vascular cambium of Arabidopsis roots, respond to cytokinins and are both required for proper root secondary thickening. Cytokinin regulation of ANT and CYCD3 also occurs during secondary thickening of poplar stems, suggesting this represents a conserved regulatory mechanism.

A molecular mechanism that confines the activity pattern of miR165 in Arabidopsis leaf primordia.

In Arabidopsis leaf primordia, the expression of HD-Zip III, which promotes tissue differentiation on the adaxial side of the leaf primordia, is repressed by miRNA165/166 (miR165/166). Small RNAs, including miRNAs, can move from cell to cell. In this study, HD-Zip III expression was strikingly repressed by miR165/166 in the epidermis and parenchyma cells on the abaxial side of the leaf primordia compared with those on the adaxial side. We also found that the MIR165A locus, which was expressed in the abaxial epidermis, was sufficient to establish the rigid repression pattern of HD-Zip III expression in the leaf primordia. Ectopic expression analyses of MIR165A showed that the abaxial-biased miR165 activity in the leaf primordia was formed neither by a polarized distribution of factors affecting miR165 activity nor by a physical boundary inhibiting the cell-to-cell movement of miRNA between the adaxial and abaxial sides. We revealed that cis-acting factors, including the promoter, backbone, and mature miRNA sequence of MIR165A, are necessary for the abaxial-biased activity of miR165 in the leaf primordia. We also found that the abaxial-determining genes YABBYs are trans-acting factors that are necessary for the miR165 activity pattern, resulting in the rigid determination of the adaxial-abaxial boundary in leaf primordia. Thus, we proposed a molecular mechanism in which the abaxial-biased patterning of miR165 activity is confined.

Plant development. Arabidopsis NAC45/86 direct sieve element morphogenesis culminating in enucleation.

Photoassimilates such as sugars are transported through phloem sieve element cells in plants. Adapted for effective transport, sieve elements develop as enucleated living cells. We used electron microscope imaging and three-dimensional reconstruction to follow sieve element morphogenesis in Arabidopsis. We show that sieve element differentiation involves enucleation, in which the nuclear contents are released and degraded in the cytoplasm at the same time as other organelles are rearranged and the cytosol is degraded. These cellular reorganizations are orchestrated by the genetically redundant NAC domain-containing transcription factors, NAC45 and NAC86 (NAC45/86). Among the NAC45/86 targets, we identified a family of genes required for enucleation that encode proteins with nuclease domains. Thus, sieve elements differentiate through a specialized autolysis mechanism.

CHOLINE TRANSPORTER-LIKE1 is required for sieve plate development to mediate long-distance cell-to-cell communication.

Phloem, a plant tissue responsible for long-distance molecular transport, harbours specific junctions, sieve areas, between the conducting cells. To date, little is known about the molecular framework related to the biogenesis of these sieve areas. Here we identify mutations at the CHER1/AtCTL1 locus of Arabidopsis thaliana. The mutations cause several phenotypic abnormalities, including reduced pore density and altered pore structure in the sieve areas associated with impaired phloem function. CHER1 encodes a member of a poorly characterized choline transporter-like protein family in plants and animals. We show that CHER1 facilitates choline transport, localizes to the trans-Golgi network, and during cytokinesis is associated with the phragmoplast. Consistent with its function in the elaboration of the sieve areas, CHER1 has a sustained, polar localization in the forming sieve plates. Our results indicate that the regulation of choline levels is crucial for phloem development and conductivity in plants.

Small RNAs as positional signal for pattern formation.

Pattern formation in plant relies on intimate cell-cell communication exchanging positional information. While ligand-receptor interaction is commonly used by plants and animals as a means to transmit positional information, plant cells can directly exchange regulatory molecules such as transcription factors through a cytoplasmic continuum called the plasmodesmata. Recently endogenous small RNAs (sRNAs) of various biogenetic origins have been shown to function non-cell-autonomously. To date, non-cell-autonomous sRNAs have been shown to regulate leaf polarity, root vascular patterning, meristem formation in embryos, shoot meristem maintenance and female gametogenesis. All these developmental processes are fundamental to the life cycle and architecture of flowering plants, suggesting that sRNA-mediated cell-to-cell signaling has been adopted to achieve novel morphology in the course of plant evolution.

Tryptophan-dependent auxin biosynthesis is required for HD-ZIP III-mediated xylem patterning.

The development and growth of higher plants is highly dependent on the conduction of water and minerals throughout the plant by xylem vessels. In Arabidopsis roots the xylem is organized as an axis of cell files with two distinct cell fates: the central metaxylem and the peripheral protoxylem. During vascular development, high and low expression levels of the class III HD-ZIP transcription factors promote metaxylem and protoxylem identities, respectively. Protoxylem specification is determined by both mobile, ground tissue-emanating miRNA165/6 species, which downregulate, and auxin concentrated by polar transport, which promotes HD-ZIP III expression. However, the factors promoting high HD-ZIP III expression for metaxylem identity have remained elusive. We show here that auxin biosynthesis promotes HD-ZIP III expression and metaxylem specification. Several auxin biosynthesis genes are expressed in the outer layers surrounding the vascular tissue in Arabidopsis root and downregulation of HD-ZIP III expression accompanied by specific defects in metaxylem development is seen in auxin biosynthesis mutants, such as trp2-12, wei8 tar2 or a quintuple yucca mutant, and in plants treated with L-kynurenine, a pharmacological inhibitor of auxin biosynthesis. Some of the patterning defects can be suppressed by synthetically elevated HD-ZIP III expression. Taken together, our results indicate that polar auxin transport, which was earlier shown to be required for protoxylem formation, is not sufficient to establish a proper xylem axis but that root-based auxin biosynthesis is additionally required.

Symplastic intercellular connectivity regulates lateral root patterning.

Cell-to-cell communication coordinates the behavior of individual cells to establish organ patterning and development. Although mobile signals are known to be important in lateral root development, the role of plasmodesmata (PD)-mediated transport in this process has not been investigated. Here, we show that changes in symplastic connectivity accompany and regulate lateral root organogenesis in Arabidopsis. This connectivity is dependent upon callose deposition around PD affecting molecular flux through the channel. Two plasmodesmal-localized ß-1,3 glucanases (PdBGs) were identified that regulate callose accumulation and the number and distribution of lateral roots. The fundamental role of PD-associated callose in this process was illustrated by the induction of similar phenotypes in lines with altered callose turnover. Our results show that regulation of callose and cell-to-cell connectivity is critical in determining the pattern of lateral root formation, which influences root architecture and optimal plant performance.

A comprehensive expression analysis of the Arabidopsis MICRORNA165/6 gene family during embryogenesis reveals a conserved role in meristem specification and a non-cell-autonomous function.

One of the most fundamental events in plant ontogeny is the specification of the shoot and root apical meristem (SAM and RAM) in embryogenesis. In Arabidopsis, the restricted expression of class III homeodomain leucine zipper (HD-ZIP III) transcription factors (TFs) at the central-apical domain of early embryos is required for the correct specification of the SAM and RAM. Because the expression of HD-ZIP III TFs is suppressed by microRNA165/166 (miR165/6), elucidation of the sites of miR165/6 production and their activity range is a key to understanding the molecular basis of SAM and RAM specification in embryogenesis. Here, we present a comprehensive reporter analysis of all nine Arabidopsis MICRORNA165/166 (MIR165/6) genes during embryogenesis. We show that five MIR165/6 genes are transcribed in a largely conserved pattern in embryos, with their expression being preferentially focused at the basal-peripheral region of embryos. Our analysis also indicated that MIR165/6 transcription does not depend on SCARECROW (SCR) function in early embryos, in contrast to its requirement in post-embryonic roots. Furthermore, by observing the expression pattern of the miR-resistant PHBmu-GFP (green fluorescent protein) reporter, in either the presence or absence of the MIR165Amu transgene, which targets PHBmu-GFP, we obtained data that indicate a non-cell-autonomous function for miR165 in early embryos. These results suggest that miR165, and possibly miR166 as well, has the capacity to act as a positional cue from the basal-peripheral region of early embryos, and remotely controls SAM and RAM specification with their non-cell-autonomous function.