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.

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.

Tissue growth constrains root organ outlines into an isometrically scalable shape.

Organ morphologies are diverse but also conserved under shared developmental constraints among species. Any geometrical similarities in the shape behind diversity and the underlying developmental constraints remain unclear. Plant root tip outlines commonly exhibit a dome shape, which likely performs physiological functions, despite the diversity in size and cellular organization among distinct root classes and/or species. We carried out morphometric analysis of the primary roots of ten angiosperm species and of the lateral roots (LRs) of Arabidopsis, and found that each root outline was isometrically scaled onto a parameter-free catenary curve, a stable structure adopted for arch bridges. Using the physical model for bridges, we analogized that localized and spatially uniform occurrence of oriented cell division and expansion force the LR primordia (LRP) tip to form a catenary curve. These growth rules for the catenary curve were verified by tissue growth simulation of developing LRP development based on time-lapse imaging. Consistently, LRP outlines of mutants compromised in these rules were found to deviate from catenary curves. Our analyses demonstrate that physics-inspired growth rules constrain plant root tips to form isometrically scalable catenary curves.

The 33rd International Conference on Arabidopsis Research (ICAR2023).

The 33rd International Conference on Arabidopsis Research (ICAR2023) was held at Makuhari Messe International Conference Hall in Chiba prefecture from June 5 to 9, 2023. This annual conference, which rotates among hosts in North America, Asia-Oceania, and Europe, covers the full range of plant biology research involving Arabidopsis and other plant species. The conference hosted more than 1200 participants, including approximately 800 international attendees from 42 countries (or regions), and featured about 900 oral and poster presentations. Reflecting the conference theme, "Arabidopsis for Sustainable Development Goals (SDGs)," there were numerous exemplary presentations regarding basic plant science using Arabidopsis and translational research conducted to achieve SDGs by exploiting the knowledge gained from Arabidopsis to improve crop production. The conference concluded on a high note, with more than 99% of survey respondents expressing their general satisfaction with ICAR2023. This report aims to summarize the organization, objectives, and outcomes of the conference.

Three-Dimensional Morphological Analysis Revealed the Cell Patterning Bases for the Sexual Dimorphism Development in the Liverwort Marchantia polymorpha.

In land plants, sexual dimorphism can develop in both diploid sporophytes and haploid gametophytes. While developmental processes of sexual dimorphism have been extensively studied in the sporophytic reproductive organs of model flowering plants such as stamens and carpels of Arabidopsis thaliana, those occurring in gametophyte generation are less well characterized due to the lack of amenable model systems. In this study, we performed three-dimensional morphological analyses of gametophytic sexual branch differentiation in the liverwort Marchantia polymorpha, using high-depth confocal imaging and a computational cell segmentation technique. Our analysis revealed that the specification of germline precursors initiates in a very early stage of sexual branch development, where incipient branch primordia are barely recognizable in the apical notch region. Moreover, spatial distribution patterns of germline precursors differ between males and females from the initial stage of primordium development in a manner dependent on the master sexual differentiation regulator MpFGMYB. At later stages, distribution patterns of germline precursors predict the sex-specific gametangia arrangement and receptacle morphologies seen in mature sexual branches. Taken together, our data suggest a tightly coupled progression of germline segregation and sexual dimorphism development in M. polymorpha.

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.

A cis-acting bidirectional transcription switch controls sexual dimorphism in the liverwort.

Plant life cycles alternate between haploid gametophytes and diploid sporophytes. While regulatory factors determining male and female sexual morphologies have been identified for sporophytic reproductive organs, such as stamens and pistils of angiosperms, those regulating sex-specific traits in the haploid gametophytes that produce male and female gametes and hence are central to plant sexual reproduction are poorly understood. Here, we identified a MYB-type transcription factor, MpFGMYB, as a key regulator of female sexual differentiation in the haploid-dominant dioicous liverwort, Marchantia polymorpha MpFGMYB is specifically expressed in females and its loss resulted in female-to-male sex conversion. Strikingly, MpFGMYB expression is suppressed in males by a cis-acting antisense gene SUF at the same locus, and loss-of-function suf mutations resulted in male-to-female sex conversion. Thus, the bidirectional transcription module at the MpFGMYB/SUF locus acts as a toggle between female and male sexual differentiation in M. polymorpha gametophytes. Arabidopsis thaliana MpFGMYB orthologs are known to be expressed in embryo sacs and promote their development. Thus, phylogenetically related MYB transcription factors regulate female gametophyte development across land plants.

CORONA, PHABULOSA and PHAVOLUTA collaborate with BELL1 to confine WUSCHEL expression to the nucellus in Arabidopsis ovules.

Angiosperm ovules consist of three proximal-distal domains - the nucellus, chalaza and funiculus - demarcated by developmental fate and specific gene expression. Mutation in three paralogous class III homeodomain leucine zipper (HD-ZIPIII) genes leads to aberrations in ovule integument development. Expression of WUSCHEL (WUS) is normally confined to the nucellar domain, but in this triple mutant expression expands into the chalaza. MicroRNA-induced suppression of this expansion partially suppresses the effects of the HD-ZIPIII mutations on ovule development, implicating ectopic WUS expression as a component of the mutant phenotype. bell1 (bel1) mutants produce aberrant structures in place of the integuments and WUS is ectopically expressed in these structures. Combination of bel1 with the HD-ZIPIII triple mutant leads to a striking phenotype in which ectopic ovules emerge from nodes of ectopic WUS expression along the funiculi of the primary ovules. The synergistic phenotype indicates that BEL1 and the HD-ZIPIII genes act in at least partial independence in confining WUS expression to the nucellus and maintaining ovule morphology. The branching ovules of the mutant resemble those of some fossil gymnosperms, implicating BEL1 and HD-ZIPIII genes as players in the evolution of the unbranched ovule form in extant angiosperms.

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.

Lateral root initiation requires the sequential induction of transcription factors LBD16 and PUCHI in Arabidopsis thaliana.

Lateral root (LR) formation in Arabidopsis thaliana is initiated by asymmetric division of founder cells, followed by coordinated cell proliferation and differentiation for patterning new primordia. The sequential developmental processes of LR formation are triggered by a localized auxin response. LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16), an auxin-inducible transcription factor, is one of the key regulators linking auxin response in LR founder cells to LR initiation. We identified key genes for LR formation that are activated by LBD16 in an auxin-dependent manner. LBD16 targets identified include the transcription factor gene PUCHI, which is required for LR primordium patterning. We demonstrate that LBD16 activity is required for the auxin-inducible expression of PUCHI. We show that PUCHI expression is initiated after the first round of asymmetric cell division of LR founder cells and that premature induction of PUCHI during the preinitiation phase disrupts LR primordium formation. Our results indicate that LR initiation requires the sequential induction of transcription factors LBD16 and PUCHI.

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.

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.

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.

The Naming of Names: Guidelines for Gene Nomenclature in Marchantia.

While Marchantia polymorpha has been utilized as a model system to investigate fundamental biological questions for over almost two centuries, there is renewed interest in M. polymorpha as a model genetic organism in the genomics era. Here we outline community guidelines for M. polymorpha gene and transgene nomenclature, and we anticipate that these guidelines will promote consistency and reduce both redundancy and confusion in the scientific literature.

Hedgehog-Gli activators direct osteo-chondrogenic function of bone morphogenetic protein toward osteogenesis in the perichondrium.

Specification of progenitors into the osteoblast lineage is an essential event for skeletogenesis. During endochondral ossification, cells in the perichondrium give rise to osteoblast precursors. Hedgehog (Hh) and bone morphogenetic protein (BMP) are suggested to regulate the commitment of these cells. However, properties of perichondrial cells and regulatory mechanisms of the specification process are still poorly understood. Here, we investigated the machineries by combining a novel organ culture system and single-cell expression analysis with mouse genetics and biochemical analyses. In a metatarsal organ culture reproducing bone collar formation, activation of BMP signaling enhanced the bone collar formation cooperatively with Hh input, whereas the signaling induced ectopic chondrocyte formation in the perichondrium without Hh input. Similar phenotypes were also observed in compound mutant mice, where signaling activities of Hh and BMP were genetically manipulated. Single-cell quantitative RT-PCR analyses showed heterogeneity of perichondrial cells in terms of natural characteristics and responsiveness to Hh input. In vitro analyses revealed that Hh signaling suppressed BMP-induced chondrogenic differentiation; Gli1 inhibited the expression of Sox5, Sox6, and Sox9 (SRY box-containing gene 9) as well as transactivation by Sox9. Indeed, ectopic expression of chondrocyte maker genes were observed in the perichondrium of metatarsals in Gli1(-/-) fetuses, and the phenotype was more severe in Gli1(-/-);Gli2(-/-) newborns. These data suggest that Hh-Gli activators alter the function of BMP to specify perichondrial cells into osteoblasts; the timing of Hh input and its target populations are critical for BMP function.

A GAL4-based targeted activation tagging system in Arabidopsis thaliana.

Activation tagging is a powerful tool for discovering novel genes that are not easily identified by loss-of-function (lof) screening due to genetic redundancy or lethality. Although the current activation tagging system, which involves a viral enhancer sequence, has been used for a decade, alternative methods that allow organ- or tissue-specific activation are required to identify genes whose strong activation leads to loss of fertility or viability. Here, we established a GAL4/UAS activation-tagging system in Arabidopsis thaliana. Host plants that express a synthetic transcription activator GAL4:VP16 (GV) in an organ- or tissue-specific manner were transformed with a T-DNA harboring tandem copies of UAS, a GAL4-binding sequence. Using a post-embryonic and root-specific GV-expressing line as the host plant, we isolated several dominant mutants with abnormal root tissue patterns, designated as uas-tagged root patterning (urp) mutants, and identified their causal genes. Notably, most URP genes encoded putative transcription factors, indicating that the GAL4/UAS activation tagging system effectively identifies genes with regulatory functions. lof phenotypes of most URP genes were either local patterning defects or visible only if homologous genes were disrupted simultaneously or independently. Systemic overexpression of some URP genes resulted in seedling lethality. These results indicate that GAL4/UAS activation tagging is a powerful method for identifying genes with biological functions that are not readily identified by conventional screening methods.

Non-cell-autonomous microRNA165 acts in a dose-dependent manner to regulate multiple differentiation status in the Arabidopsis root.

In the development of multicellular organisms, cell fate is usually determined by exchanging positional information. Animals employ a class of intercellular signaling molecules that specify different cell fates by their dosage, but the existence of an equivalent system has not been demonstrated in plants, except that the growth regulator auxin has been proposed to act in a similar manner in certain developmental contexts. Recently, it has been reported that, in the Arabidopsis root meristem, endodermis-derived microRNA (miR) 165/166 non-cell-autonomously suppress the expression of the Class III HD-ZIP transcription factor PHABULOSA (PHB) in the peripheral stele, thereby specifying xylem differentiation. Here, we show that the miR165/166-dependent suppression of PHB is required not only for xylem specification, but also for differentiation of the pericycle, as well as for ground tissue patterning. Furthermore, using a plant system that allows quantitative control of miR165 production in the ground tissue, we show that endodermis-derived miR165 acts in a dose-dependent manner to form a graded distribution of PHB transcripts across the stele. These results reveal a previously unidentified role of miR165 in the differentiation of a broad range of root cell types and suggest that endodermis-derived miR165 acts in a dose-dependent manner to control multiple differentiation status in the Arabidopsis root.

Gli1 protein participates in Hedgehog-mediated specification of osteoblast lineage during endochondral ossification.

With regard to Hedgehog signaling in mammalian development, the majority of research has focused on Gli2 and Gli3 rather than Gli1. This is because Gli1(-/-) mice do not show any gross abnormalities in adulthood, and no detailed analyses of fetal Gli1(-/-) mice are available. In this study, we investigated the physiological role of Gli1 in osteogenesis. Histological analyses revealed that bone formation was impaired in Gli1(-/-) fetuses compared with WT fetuses. Gli1(-/-) perichondrial cells expressed neither runt-related transcription factor 2 (Runx2) nor osterix, master regulators of osteogenesis, in contrast to WT cells. In vitro analyses showed that overexpression of Gli1 up-regulated early osteogenesis-related genes in both WT and Runx2(-/-) perichondrial cells, and Gli1 activated transcription of those genes via its association with their 5'-regulatory regions, underlying the function of Gli1 in the perichondrium. Moreover, Gli1(-/-);Gli2(-/-) mice showed more severe phenotypes of impaired bone formation than either Gli1(-/-) or Gli2(-/-) mice, and osteoblast differentiation was impaired in Gli1(-/-);Gli3(-/-) perichondrial cells compared with Gli3(-/-) cells in vitro. These data suggest that Gli1 itself can induce early osteoblast differentiation, at least to some extent, in a Runx2-independent manner. It also plays a redundant role with Gli2 and is involved in the repressor function of Gli3 in osteogenesis. On the basis of these findings, we propose that upon Hedgehog input, Gli1 functions collectively with Gli2 and Gli3 in osteogenesis.

Peptide separation methodologies for in-depth proteomics in Arabidopsis.

In the post-genome era, several tools that have increased our global understanding of the molecular basis of several cell-based phenomena have been developed. However, proteomics has not been efficiently integrated with the other 'omics' (e.g. transcriptomics and metabolomics), because of the relatively low number of proteins identified by mass spectrometry (MS). Peptides from low-abundance proteins are often not detected by MS due to ionization suppression. To improve the number of peptide identifications in MS analyses, we propose three separation methodologies; namely, OFFGEL electrophoresis, 2D-liquid chromatography (LC) and the long monolithic silica-C18 capillary column method, with the common aim to decrease peptide complexity prior to MS analyses. Proteomics using the above three peptide separation methods were separately applied to protoplasts collected from the epidermal cell layer of Arabidopsis roots using fluorescence-activated cell sorting. In each method alone, 1,132, 836 and 795 proteins were specifically identified, respectively. This has allowed the identification of 1,493 proteins with no redundancy and with <1.0% false discovery rate. Moreover, approximately two-thirds of these proteins are identified here for the first time in the epidermal cell layer. These results show that use of different proteomic approaches can increase the total number of proteins identified. We propose that the integration of data from these methodologies represents a powerful tool for generation of proteome maps by enabling identification of low-abundance proteins in the various Arabidopsis root cell layers.