Plasmodesmata dynamics in bryophyte model organisms: secondary formation and developmental modifications of structure and function.

MAIN CONCLUSION: Developing bryophytes differentially modify their plasmodesmata structure and function. Secondary plasmodesmata formation via twinning appears to be an ancestral trait. Plasmodesmata networks in hornwort sporophyte meristems resemble those of angiosperms. All land-plant taxa use plasmodesmata (PD) cell connections for symplasmic communication. In angiosperm development, PD networks undergo an extensive remodeling by structural and functional PD modifications, and by postcytokinetic formation of additional secondary PD (secPD). Since comparable information on PD dynamics is scarce for the embryophyte sister groups, we investigated maturating tissues of Anthoceros agrestis (hornwort), Physcomitrium patens (moss), and Marchantia polymorpha (liverwort). As in angiosperms, quantitative electron microscopy revealed secPD formation via twinning in gametophytes of all model bryophytes, which gives rise to laterally adjacent PD pairs or to complex branched PD. This finding suggests that PD twinning is an ancient evolutionary mechanism to adjust PD numbers during wall expansion. Moreover, all bryophyte gametophytes modify their existing PD via taxon-specific strategies resembling those of angiosperms. Development of type II-like PD morphotypes with enlarged diameters or formation of pit pairs might be required to maintain PD transport rates during wall thickening. Similar to angiosperm leaves, fluorescence redistribution after photobleaching revealed a considerable reduction of the PD permeability in maturating P. patens phyllids. In contrast to previous reports on monoplex meristems of bryophyte gametophytes with single initials, we observed targeted secPD formation in the multi-initial basal meristems of A. agrestis sporophytes. Their PD networks share typical features of multi-initial angiosperm meristems, which may hint at a putative homologous origin. We also discuss that monoplex and multi-initial meristems may require distinct types of PD networks, with or without secPD formation, to control maintenance of initial identity and positional signaling.

The role of D3-type cyclins is related to cytokinin and the bHLH transcription factor SPATULA in Arabidopsis gynoecium development.

MAIN CONCLUSION: We studied the D3-type cyclin function during gynoecium development in Arabidopsis and how they are related to the hormone cytokinin and the transcription factor SPATULA. Growth throughout the life of plants is sustained by cell division and differentiation processes in meristematic tissues. In Arabidopsis, gynoecium development implies a multiphasic process where the tissues required for pollination, fertilization, and seed development form. The Carpel Margin Meristem (CMM) is a mass of undifferentiated cells that gives rise to the gynoecium internal tissues, such as septum, ovules, placenta, funiculus, transmitting tract, style, and stigma. Different genetic and hormonal factors, including cytokinin, control the CMM function. Cytokinin regulates the cell cycle transitions through the activation of cell cycle regulators as cyclin genes. D3-type cyclins are expressed in proliferative tissues, favoring the mitotic cell cycle over the endoreduplication. Though the role of cytokinin in CMM and gynoecium development is highly studied, its specific role in regulating the cell cycle in this tissue remains unclear. Additionally, despite extensive research on the relationship between CYCD3 genes and cytokinin, the regulatory mechanism that connects them remains elusive. Here, we found that D3-type cyclins are expressed in proliferative medial and lateral tissues. Conversely, the depletion of the three CYCD3 genes showed that they are not essential for gynoecium development. However, the addition of exogenous cytokinin showed that they could control the division/differentiation balance in gynoecium internal tissues and outgrowths. Finally, we found that SPATULA can be a mechanistic link between cytokinin and the D3-type cyclins. The data suggest that the role of D3-type cyclins in gynoecium development is related to the cytokinin response, and they might be activated by the transcription factor SPATULA.

Uncovering PheCLE1 and PheCLE10 Promoting Root Development Based on Genome-Wide Analysis.

Moso bamboo (Phyllostachys edulis), renowned for its rapid growth, is attributed to the dynamic changes in its apical meristem. The CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) family genes are known to play crucial roles in regulating meristem and organ formation in model plants, but their functions in Moso bamboo remain unclear. Here, we conducted a genome-wide identification of the CLE gene family of Moso bamboo and investigated their gene structure, chromosomal localization, evolutionary relationships, and expression patterns. A total of 11 PheCLE genes were identified, all of which contained a conserved CLE peptide core functional motif (Motif 1) at their C-termini. Based on Arabidopsis classification criteria, these genes were predominantly distributed in Groups A-C. Collinearity analysis unveiled significant synteny among CLE genes in Moso bamboo, rice, and maize, implying potential functional conservation during monocot evolution. Transcriptomic analysis showed significant expression of these genes in the apical tissues of Moso bamboo, including root tips, shoot tips, rhizome buds, and flower buds. Particularly, single-cell transcriptomic data and in situ hybridization further corroborated the heightened expression of PheCLE1 and PheCLE10 in the apical tissue of basal roots. Additionally, the overexpression of PheCLE1 and PheCLE10 in rice markedly promoted root growth. PheCLE1 and PheCLE10 were both located on the cell membrane. Furthermore, the upstream transcription factors NAC9 and NAC6 exhibited binding affinity toward the promoters of PheCLE1 and PheCLE10, thereby facilitating their transcriptional activation. In summary, this study not only systematically identified the CLE gene family in Moso bamboo for the first time but also emphasized their central roles in apical tissue development. This provides a valuable theoretical foundation for the further exploration of functional peptides and their signaling regulatory networks in bamboo species.

In Planta Genome Editing in Commercial Wheat Varieties: Use of TaQsd1 to Lengthen Seed Dormancy.

Dependency on in vitro culture and regeneration limits the ability to use genome editing on elite wheat (Triticum aestivum L.) varieties. We recently developed an in planta particle bombardment (iPB) technique for gene editing in wheat that utilizes shoot apical meristems (SAMs) as a target tissue. Since the method does not require in vitro culture, it can therefore be used on recalcitrant varieties. In this chapter, we describe in detail the steps used in the iPB method. With this protocol, 3% to 5% of T0 plants grown from bombarded SAMs typically carry mutant alleles and approximately 1% to 2% of the T0 plants inherit mutant alleles in the next generation.

Seed production determines the entrance to dormancy of the inflorescence meristem of Pisum sativum and the end of the flowering period.

Flowering plants adjust their reproductive period to maximize the success of the offspring. Monocarpic plants, those with a single reproductive cycle that precedes plant senescence and death, tightly regulate both flowering initiation and flowering cessation. The end of the flowering period involves the arrest of the inflorescence meristem activity, known as proliferative arrest, in what has been interpreted as an evolutionary adaptation to maximize the allocation of resources to seed production and the viability of the progeny. Factors influencing proliferative arrest were described for several monocarpic plant species many decades ago, but only in the last few years studies performed in Arabidopsis have allowed to approach proliferative arrest regulation in a comprehensive manner by studying the physiology, hormone dynamics, and genetic factors involved in its regulation. However, these studies remain restricted to Arabidopsis and there is a need to expand our knowledge to other monocarpic species to propose general mechanisms controlling the process. In this work, we have characterized proliferative arrest in Pisum sativum, trying to parallel available studies in Arabidopsis to maximize this comparative framework. We have assessed quantitatively the role of fruits/seeds in the process, the influence of the positional effect of these fruits/seeds in the behavior of the inflorescence meristem, and the transcriptomic changes in the inflorescence associated with the arrested state of the meristem. Our results support a high conservation of the factors triggering arrest in pea and Arabidopsis, but also reveal differences reinforcing the need to perform similar studies in other species.

Transcriptional response of Arabidopsis thaliana's root-tip to spaceflight.

Plants are expected to play a critical role in the biological life support systems of crewed spaceflight missions, including in the context of upcoming missions targeting the Moon and Mars. Therefore, understanding the response of plants to spaceflight is essential for improving the selection and engineering of plants and spaceflight conditions. In particular, understanding the root-tip's response to spaceflight is of importance as it is the center of orchestrating the development of the root, the primary organ for the absorption of nutrients and anchorage. GLDS-120 is a pioneering study by Paul et al. that used transcriptomics to evaluate the spaceflight response of the root-tip of the model plant Arabidopsis thaliana in dark and light through separate analyses of three genotype groups (Wassilewskija, Columbia-0, and Columbia-0 PhyD) and comparison of genotype responses. Here, we provide a complementary analysis of this dataset through a combined analysis of all samples while controlling for the genotypes in a paired analysis. We identified a robust transcriptional response to spaceflight with 622 DEGs in light and 200 DEGs in dark conditions. Gene enrichment analysis identified 37 and 13 significantly enriched terms from biological processes in light and dark conditions, respectively. Prominent enrichment for hypoxia-related terms in both conditions suggests hypoxia is a key stressor for root development during spaceflight. Additional enriched terms in light conditions include the circadian cycle, light response, and terms for the metabolism of flavonoid and indole-containing compounds. These results further our understanding of plants' responses to the spaceflight environment.

YABBY and diverged KNOX1 genes shape nodes and internodes in the stem.

Plant stems comprise nodes and internodes that specialize in solute exchange and elongation. However, their boundaries are not well defined, and how these basic units arise remains elusive. In rice with clear nodes and internodes, we found that one subclade of class I knotted1-like homeobox (KNOX1) genes for shoot meristem indeterminacy restricts node differentiation and allows internode formation by repressing YABBY genes for leaf development and genes from another node-specific KNOX1 subclade. YABBYs promote nodal vascular differentiation and limit stem elongation. YABBY and node-specific KNOX1 genes specify the pulvinus, which further elaborates the nodal structure for gravitropism. Notably, this KNOX1 subclade organization is specific to seed plants. We propose that nodes and internodes are distinct domains specified by YABBY-KNOX1 cross-regulation that diverged in early seed plants.

The people behind the papers - Léa Rambaud-Lavigne, Namrata Gundiah, Arezki Boudaoud and Pradeep Das.

The shoot apical meristem is a key stem cell niche in plants, and proper stem cell maintenance is partly regulated by CLAVATA3 (CLV3). Without CLV3 meristems overgrow, but the mechanistic basis of this phenotype was unclear. A new paper in Development suggests that CLV3 modulates the physical properties of meristematic stem cells, and that these properties help shape meristem morphology. To learn more about the story behind the paper, we caught up with first author Léa Rambaud-Lavigne and corresponding authors Namrata Gundiah, Arezki Boudaoud and Pradeep Das.

Male Germ Cell Specification in Plants.

Germ cells (GCs) serve as indispensable carriers in both animals and plants, ensuring genetic continuity across generations. While it is generally acknowledged that the timing of germline segregation differs significantly between animals and plants, ongoing debates persist as new evidence continues to emerge. In this review, we delve into studies focusing on male germ cell specifications in plants, and we summarize the core gene regulatory circuits in germ cell specification, which show remarkable parallels to those governing meristem homeostasis. The similarity in germline establishment between animals and plants is also discussed.

Progressive meristem and single-cell transcriptomes reveal the regulatory mechanisms underlying maize inflorescence development and sex differentiation.

Maize develops separate ear and tassel inflorescences with initially similar morphology but ultimately different architecture and sexuality. The detailed regulatory mechanisms underlying these changes still remain largely unclear. In this study, through analyzing the time-course meristem transcriptomes and floret single-cell transcriptomes of ear and tassel, we revealed the regulatory dynamics and pathways underlying inflorescence development and sex differentiation. We identified 16 diverse gene clusters with differential spatiotemporal expression patterns and revealed biased regulation of redox, programmed cell death, and hormone signals during meristem differentiation between ear and tassel. Notably, based on their dynamic expression patterns, we revealed the roles of two RNA-binding proteins in regulating inflorescence meristem activity and axillary meristem formation. Moreover, using the transcriptional profiles of 53 910 single cells, we uncovered the cellular heterogeneity between ear and tassel florets. We found that multiple signals associated with either enhanced cell death or reduced growth are responsible for tassel pistil suppression, while part of the gibberellic acid signal may act non-cell-autonomously to regulate ear stamen arrest during sex differentiation. We further showed that the pistil-protection gene SILKLESS 1 (SK1) functions antagonistically to the known pistil-suppression genes through regulating common molecular pathways, and constructed a regulatory network for pistil-fate determination. Collectively, our study provides a deep understanding of the regulatory mechanisms underlying inflorescence development and sex differentiation in maize, laying the foundation for identifying new regulators and pathways for maize hybrid breeding and improvement.

Vascular cambium stem cells: past, present and future.

Secondary xylem and phloem originate from a lateral meristem called the vascular cambium that consists of one to several layers of meristematic cells. Recent lineage tracing studies have shown that only one of the cambial cells in each radial cell file functions as the stem cell, capable of producing both secondary xylem and phloem. Here, we first review how phytohormones and signalling peptides regulate vascular cambium formation and activity. We then propose how the stem cell concept, familiar from apical meristems, could be applied to cambium studies. Finally, we discuss how this concept could set the basis for future research.

BdRCN4, a Brachypodium distachyon TFL1 homologue, is involved in regulation of apical meristem fate.

In higher plants, the shift from vegetative to reproductive development is governed by complex interplay of internal and external signals. TERMINALFLOWER1 (TFL1) plays a crucial role in the regulation of flowering time and inflorescence architecture in Arabidopsis thaliana. This study aimed to explore the function of BdRCN4, a homolog of TFL1 in Brachypodium distachyon, through functional analyses in mutant and transgenic plants. The results revealed that overexpression of BdRCN4 in B. distachyon leads to an extended vegetative phase and reduced production of spikelets. Similar results were found in A. thaliana, where constitutive expression of BdRCN4 promoted a delay in flowering time, followed by the development of hypervegetative shoots, with no flowers or siliques produced. Our results suggest that BdRCN4 acts as a flowering repressor analogous to TFL1, negatively regulating AP1, but no LFY expression. To further validate this hypothesis, a 35S::LFY-GR co-transformation approach on 35::BdRCN4 lines was performed. Remarkably, AP1 expression levels and flower formation were restored to normal in co-transformed plants when treated with dexamethasone. Although further molecular studies will be necessary, the evidence in B. distachyon support the idea that a balance between LFY and BdRCN4/TFL1 seems to be essential for activating AP1 expression and initiating floral organ identity gene expression. This study also demonstrates interesting conservation through the molecular pathways that regulate flowering meristem transition and identity across the evolution of monocot and dicot plants.

PLETHORA transcription factors promote early embryo development through induction of meristematic potential.

Plants are dependent on divisions of stem cells to establish cell lineages required for growth. During embryogenesis, early division products are considered to be stem cells, whereas during post-embryonic development, stem cells are present in meristems at the root and shoot apex. PLETHORA/AINTEGUMENTA-LIKE (PLT/AIL) transcription factors are regulators of post-embryonic meristem function and are required to maintain stem cell pools. Despite the parallels between embryonic and post-embryonic stem cells, the role of PLTs during early embryogenesis has not been thoroughly investigated. Here, we demonstrate that the PLT regulome in the zygote, and apical and basal cells is in strong congruence with that of post-embryonic meristematic cells. We reveal that out of all six PLTs, only PLT2 and PLT4/BABY BOOM (BBM) are expressed in the zygote, and that these two factors are essential for progression of embryogenesis beyond the zygote stage and first divisions. Finally, we show that other PLTs can rescue plt2 bbm defects when expressed from the PLT2 and BBM promoters, establishing upstream regulation as a key factor in early embryogenesis. Our data indicate that generic PLT factors facilitate early embryo development in Arabidopsis by induction of meristematic potential.

CDC48A, an interactor of WOX2, is required for embryonic patterning in Arabidopsis thaliana.

KEY MESSAGE: Interactor of WOX2, CDC48A, is crucial for early embryo patterning and shoot meristem stem cell initiation, but is not required for WOX2 protein turnover or subcellular localization. During Arabidopsis embryo patterning, the WUSCHEL HOMEOBOX 2 (WOX2) transcription factor is a major regulator of protoderm and shoot stem cell initiation. Loss of WOX2 function results in aberrant protodermal cell divisions and, redundantly with its paralogs WOX1, WOX3, and WOX5, compromised shoot meristem formation. To elucidate the molecular basis for WOX2 function, we searched for protein interactors by IP-MS/MS from WOX2-overexpression roots displaying reprogramming toward shoot-like cell fates. Here, we report that WOX2 directly interacts with the type II AAA ATPase molecular chaperone CELL DIVISION CYCLE 48A (CDC48A). We confirmed this interaction with bimolecular fluorescence complementation and co-immunoprecipitation and found that both proteins co-localize in the nucleus. We show that CDC48A loss of function results in protoderm and shoot meristem stem cell initiation defects similar to WOX2 loss of function. We also provide evidence that CDC48A promotes WOX2 activity independently of proteolysis or the regulation of nuclear localization, common mechanisms of CDC48A function in other processes. Our results point to a new role of CDC48A in potentiating WOX2 function during early embryo patterning.

Protocol to efficiently induce CRISPR-Kill-mediated cell ablation in Arabidopsis thaliana.

The CRISPR-Kill system enables targeted cell ablation by inducing multiple double-strand breaks in evolutionarily conserved repetitive genomic regions. Here, we present a protocol for the application of the CRISPR-Kill system to analyze the systemic and cellular effects of targeted cell death in Arabidopsis. We describe steps for generating constitutive and inducible CRISPR-Kill lines, chemically inducing CRISPR-Cas9-mediated genome elimination, and monitoring of cell death in shoot and root apical meristems. This enables the investigation of a wide range of questions in developmental plant biology. For complete details on the use and execution of this protocol, please refer to Gehrke et al.1.

Tuber, or not tuber: Molecular and morphological basis of underground storage organ development.

Underground storage organs occur in phylogenetically diverse plant taxa and arise from multiple tissue types including roots and stems. Thickening growth allows underground storage organs to accommodate carbohydrates and other nutrients and requires proliferation at various lateral meristems followed by cell expansion. The WOX-CLE module regulates thickening growth via the vascular cambium in several eudicot systems, but the molecular mechanisms of proliferation at other lateral meristems are not well understood. In potato, onion, and other systems, members of the phosphatidylethanolamine-binding protein (PEBP) gene family induce underground storage organ development in response to photoperiod cues. While molecular mechanisms of tuber development in potato are well understood, we lack detailed mechanistic knowledge for the extensive morphological and taxonomic diversity of underground storage organs in plants.

Arabidopsis AN3 and OLIGOCELLULA genes link telomere maintenance mechanisms with cell division and expansion control.

Telomeres are conserved chromosomal structures necessary for continued cell division and proliferation. In addition to the classical telomerase pathway, multiple other genes including those involved in ribosome metabolism and chromatin modification contribute to telomere length maintenance. We previously reported that Arabidopsis thaliana ribosome biogenesis genes OLI2/NOP2A, OLI5/RPL5A and OLI7/RPL5B have critical roles in telomere length regulation. These three OLIGOCELLULA genes were also shown to function in cell proliferation and expansion control and to genetically interact with the transcriptional co-activator ANGUSTIFOLIA3 (AN3). Here we show that AN3-deficient plants progressively lose telomeric DNA in early homozygous mutant generations, but ultimately establish a new shorter telomere length setpoint by the fifth mutant generation with a telomere length similar to oli2/nop2a -deficient plants. Analysis of double an3 oli2 mutants indicates that the two genes are epistatic for telomere length control. Telomere shortening in an3 and oli mutants is not caused by telomerase inhibition; wild type levels of telomerase activity are detected in all analyzed mutants in vitro. Late generations of an3 and oli mutants are prone to stem cell damage in the root apical meristem, implying that genes regulating telomere length may have conserved functional roles in stem cell maintenance mechanisms. Multiple instances of anaphase fusions in late generations of oli5 and oli7 mutants were observed, highlighting an unexpected effect of ribosome biogenesis factors on chromosome integrity. Overall, our data implicate AN3 transcription coactivator and OLIGOCELLULA proteins in the establishment of telomere length set point in plants and further suggest that multiple regulators with pleiotropic functions can connect telomere biology with cell proliferation and cell expansion pathways.

The peptidase DA1 cleaves and destabilizes WUSCHEL to control shoot apical meristem size.

Stem cells in plants and animals are the source of new tissues and organs. In plants, stem cells are maintained in the central zone (CZ) of multicellular meristems, and large shoot meristems with an increased stem cell population hold promise for enhancing yield. The mobile homeodomain transcription factor WUSCHEL (WUS) is a central regulator of stem cell function in plant shoot meristems. Despite its central importance, the factors that directly modulate WUS protein stability have been a long-standing question. Here, we show that the peptidase DA1 physically interacts with and cleaves the WUS protein, leading to its destabilization. Furthermore, our results reveal that cytokinin signaling represses the level of DA1 protein in the shoot apical meristem, thereby increasing the accumulation of WUS protein. Consistent with these observations, loss of DA1 function results in larger shoot apical meristems with an increased stem cell population and also influences cytokinin-induced enlargement of shoot apical meristem. Collectively, our findings uncover a previously unrecognized mechanism by which the repression of DA1 by cytokinin signaling stabilizes WUS, resulting in the enlarged shoot apical meristems with the increased stem cell number during plant growth and development.

Heterogeneous identity, stiffness and growth characterise the shoot apex of Arabidopsis stem cell mutants.

Stem cell homeostasis in the shoot apical meristem involves a core regulatory feedback loop between the signalling peptide CLAVATA3 (CLV3), produced in stem cells, and the transcription factor WUSCHEL, expressed in the underlying organising centre. clv3 mutant meristems display massive overgrowth, which is thought to be caused by stem cell overproliferation, although it is unknown how uncontrolled stem cell divisions lead to this altered morphology. Here, we reveal local buckling defects in mutant meristems, and use analytical models to show how mechanical properties and growth rates may contribute to the phenotype. Indeed, clv3 mutant meristems are mechanically more heterogeneous than the wild type, and also display regional growth heterogeneities. Furthermore, stereotypical wild-type meristem organisation, in which cells simultaneously express distinct fate markers, is lost in mutants. Finally, cells in mutant meristems are auxin responsive, suggesting that they are functionally distinguishable from wild-type stem cells. Thus, all benchmarks show that clv3 mutant meristem cells are different from wild-type stem cells, suggesting that overgrowth is caused by the disruption of a more complex regulatory framework that maintains distinct genetic and functional domains in the meristem.

Visualizing 13C-Labeled Metabolites in Maize Root Tips with Mass Spectrometry Imaging.

Tracing in vivo isotope-labeled metabolites has been used to study metabolic pathways or flux analysis. However, metabolic differences between the cells have been often ignored in these studies due to the limitation of solvent-based extraction. Here we demonstrate that the mass spectrometry imaging of in vivo isotope-labeled metabolites, referred to as MSIi, can provide important insights into metabolic dynamics with cellular resolution that may supplement the traditional metabolomics and flux analysis. Developing maize root tips are adopted as a model system for MSIi by supplementing 200 mM [U-13C]glucose in 0.1x Hoagland medium. MSIi data sets were acquired for longitudinal sections of newly grown maize root tips after growing 5 days in the medium. A total of 56 metabolite features were determined to have been 13C-labeled based on accurate mass and the number of carbon matching with the metabolite databases. Simple sugars and their derivatives were fully labeled, but some small metabolites were partially labeled with a significant amount of fully unlabeled metabolites still present, suggesting the recycling of "old" metabolites in the newly grown tissues. Some distinct localizations were found, including the low abundance of hexose and its derivatives in the meristem, the high abundance of amino acids in the meristem, and the localization to epidermal and endodermal cells for lipids and their intermediates. Fatty acids and lipids were slow in metabolic turnover and showed various isotopologue distributions with intermediate building blocks, which may provide flux information for their biosynthesis.