Actin-mediated avoidance of tricellular junction influences global topology at the Arabidopsis shoot apical meristem.

Division plane orientation contributes to cell shape and topological organization, playing a key role in morphogenesis, but the precise physical and molecular mechanism influencing these processes remains largely obscure in plants. In particular, it is less clear how the placement of the new walls occurs in relation to the walls of neighboring cells. Here, we show that genetic perturbation of the actin cytoskeleton results in more rectangular cell shapes and higher incidences of four-way junctions, perturbing the global topology of cells in the shoot apical meristem of Arabidopsis thaliana. Actin mutants also exhibit changes in the expansion rate of the new versus the maternal cell wall after division, affecting the evolution of internal angles at tricellular junctions. Further, the increased width of the preprophase band in the actin mutant contributes to inaccuracy in the placement of the new cell wall. Computational simulation further substantiates this hypothesis and reproduces the observed cell shape defects.

Sugar signaling modulates SHOOT MERISTEMLESS expression and meristem function in Arabidopsis.

In plants, development of all above-ground tissues relies on the shoot apical meristem (SAM) which balances cell proliferation and differentiation to allow life-long growth. To maximize fitness and survival, meristem activity is adjusted to the prevailing conditions through a poorly understood integration of developmental signals with environmental and nutritional information. Here, we show that sugar signals influence SAM function by altering the protein levels of SHOOT MERISTEMLESS (STM), a key regulator of meristem maintenance. STM is less abundant in inflorescence meristems with lower sugar content, resulting from plants being grown or treated under limiting light conditions. Additionally, sucrose but not light is sufficient to sustain STM accumulation in excised inflorescences. Plants overexpressing the α1-subunit of SUCROSE-NON-FERMENTING1-RELATED KINASE 1 (SnRK1) accumulate less STM protein under optimal light conditions, despite higher sugar accumulation in the meristem. Furthermore, SnRK1α1 interacts physically with STM and inhibits its activity in reporter assays, suggesting that SnRK1 represses STM protein function. Contrasting the absence of growth defects in SnRK1α1 overexpressors, silencing SnRK1α in the SAM leads to meristem dysfunction and severe developmental phenotypes. This is accompanied by reduced STM transcript levels, suggesting indirect effects on STM. Altogether, we demonstrate that sugars promote STM accumulation and that the SnRK1 sugar sensor plays a dual role in the SAM, limiting STM function under unfavorable conditions but being required for overall meristem organization and integrity under favorable conditions. This highlights the importance of sugars and SnRK1 signaling for the proper coordination of meristem activities.

The WOX9-WUS modules are indispensable for the maintenance of stem cell homeostasis in Arabidopsis thaliana.

The dynamic balance between the self-renewal and differentiation of stem cells in plants is precisely regulated by a series of developmental regulated genes that exhibit spatiotemporal-specific expression patterns. Several studies have demonstrated that the WOX family transcription factors play critical roles in maintaining the identity of stem cells in Arabidopsis thaliana. In this study, we obtained amiR-WOX9 transgenic plants, which displayed terminating prematurely of shoot apical meristem (SAM) development, along with alterations in inflorescence meristem and flower development. The phenotype of amiR-WOX9 plants exhibited similarities to that of wus-101 mutant, characterized by a stop-and-go growth pattern. It was also found that the expression of WUS in amiR-WOX9 lines was decreased significantly, while in UBQ10::WOX9-GFP transgenic plants, the WUS expression was increased significantly despite no substantial alteration in meristem size compared to Col. Therefore, these data substantiated the indispensable role of WOX9 in maintaining the proper expression of WUS. Further investigations unveiled the direct binding of WOX9 to the WUS promoter via the TAAT motif, thereby activating its expression. It was also found that WUS recognized identical the same TAAT motif cis-elements in its own promoter, thereby repress self-expression. Next, we successfully identified a physical interaction between WOX9 and WUS, and verified that it was harmful to the expression of WUS. Finally, our experimental findings demonstrate that WOX9 was responsible for the direct activating of WUS, which however was interfered by the ways of WUS binding its own promoter and the interaction of WUS and WOX9, thereby ensuring the appropriate expression pattern of WUS and then the stem cell stability. This study contributes to an enhanced comprehension of the regulatory network of the WOX9-WUS module in maintaining the equilibrium of the SAM.

A dynamic WUSCHEL/Layer 1 interplay directs shoot apical meristem formation during regeneration in tobacco.

De novo shoot apical meristem (SAM) organogenesis during regeneration in tissue culture has been investigated for several decades, but the precise mechanisms governing early-stage cell fate specification remain elusive. In contrast to SAM establishment during embryogenesis, in vitro SAM formation occurs without positional cues and is characterized by autonomous initiation of cellular patterning. Here, we report on the initial stages of SAM organogenesis and on the molecular mechanisms that orchestrate gene patterning to establish SAM homeostasis. We found that SAM organogenesis in tobacco calli starts with protuberance formation followed by the formation of an intact L1 layer covering the nascent protuberance. We also exposed a complex interdependent relationship between L1 and WUS expression and revealed that any disruption in this interplay compromises shoot formation. Silencing WUS in nascent protuberances prevented L1 formation and caused the disorganization of the outer cell layers exhibiting both anticlinal and periclinal divisions, suggesting WUS plays a critical role in the proper establishment and organization of L1 during SAM organogenesis. We further discovered that silencing TONNEAU1 prevents the exclusive occurrence of anticlinal divisions in the outermost layer of the protuberances and suppresses the acquisition of L1 cellular identity and L1 formation, ultimately impeding SAM formation and regeneration. This study provides a novel molecular framework for the characterization of a WUS/L1 interplay that mediates SAM formation during regeneration.

Live Imaging of the Shoot Apical Meristem of Intact, Soil-Grown, Flowering Arabidopsis Plants.

All aerial organs in plants originate from the shoot apical meristem, a specialized tissue at the tip of a plant, enclosing a few stem cells. Understanding developmental dynamics within this tissue in relation to internal and external stimuli is of crucial importance. Imaging the meristem at the cellular level beyond very early stages requires the apex to be detached from the plant body, a procedure that does not allow studies in living, intact plants over longer periods. This protocol describes a new confocal microscopy method with the potential to image the shoot apical meristem of an intact, soil-grown, flowering Arabidopsis plant over several days. The setup opens new avenues to study apical stem cells, their interconnection with the whole plant, and their responses to environmental stimuli. Key features • Novel dissection and imaging method of the shoot apical meristem of Arabidopsis. • Procedure performed with intact, soil-grown, flowering plants. • Possibility of long-term live imaging of the shoot apical meristem. • Protocol can be adapted to different plant 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.

Antiviral defense in plant stem cells.

Undifferentiated plant and animal stem cells are essential for cell, tissue, and organ differentiation, development, and growth. They possess unusual antiviral immunity which differs from that in specialized cells. By comparison to animal stem cells, we discuss how plant stem cells defend against viral invasion and beyond.

Stem cell quiescence and dormancy in plant meristems.

Plants exhibit opportunistic developmental patterns, alternating between growth and dormancy in response to external cues. Moreover, quiescence plays a critical role in proper plant growth and development, particularly within the root apical meristem and the shoot apical meristem. In these meristematic tissues, cells with relatively slower mitotic activity are present in the quiescent center and the central zone, respectively. These centers form long-term reservoirs of stem cells maintaining the meristematic stem cell niche, and thus sustaining continuous plant development and adaptation to changing environments. This review explores early observations, structural characteristics, functions, and gene regulatory networks of the root and shoot apical meristems. It also highlights the intricate mechanism of dormancy within the shoot apical meristem. The aim is to contribute to a holistic understanding of quiescence in plants, which is fundamental for the proper growth and environmental response of plants.

An updated model of shoot apical meristem regulation by ERECTA family and CLAVATA3 signaling pathways in Arabidopsis.

The shoot apical meristem (SAM) gives rise to the aboveground organs of plants. The size of the SAM is relatively constant due to the balance between stem cell replenishment and cell recruitment into new organs. In angiosperms, the transcription factor WUSCHEL (WUS) promotes stem cell proliferation in the central zone of the SAM. WUS forms a negative feedback loop with a signaling pathway activated by CLAVATA3 (CLV3). In the periphery of the SAM, the ERECTA family receptors (ERfs) constrain WUS and CLV3 expression. Here, we show that four ligands of ERfs redundantly inhibit the expression of these two genes. Transcriptome analysis confirmed that WUS and CLV3 are the main targets of ERf signaling and uncovered new ones. Analysis of promoter reporters indicated that the WUS expression domain mostly overlaps with the CLV3 domain and does not shift along the apical-basal axis in clv3 mutants. Our three-dimensional mathematical model captured gene expression distributions at the single-cell level under various perturbed conditions. Based on our findings, CLV3 regulates cellular levels of WUS mostly through autocrine signaling, and ERfs regulate the spatial expression of WUS, preventing its encroachment into the peripheral zone.

Somatic epigenetic drift during shoot branching: a cell lineage-based model.

Plant architecture is shaped by the production of new organs, most of which emerge postembryonically. This process includes the formation of new lateral branches along existing shoots. Current evidence supports a detached-meristem model as the cellular basis of lateral shoot initiation. In this model, a small number of undifferentiated cells are sampled from the periphery of the shoot apical meristem (SAM) to act as precursors for axillary buds, which eventually develop into new shoots. Repeated branching thus creates cellular bottlenecks (i.e. somatic drift) that affect how de novo (epi)genetic mutations propagate through the plant body during development. Somatic drift could be particularly relevant for stochastic DNA methylation gains and losses (i.e. spontaneous epimutations), as they have been shown to arise rapidly with each cell division. Here, we formalize a special case of the detached-meristem model, where precursor cells are randomly sampled from the SAM periphery in a way that maximizes cell lineage independence. We show that somatic drift during repeated branching gives rise to a mixture of cellular phylogenies within the SAM over time. This process is dependent on the number of branch points, the strength of drift as well as the epimutation rate. Our model predicts that cell-to-cell DNA methylation heterogeneity in the SAM converges to nonzero states during development, suggesting that epigenetic variation is an inherent property of the SAM cell population. Our insights have direct implications for empirical studies of somatic (epi)genomic diversity in long-lived perennial and clonal species using bulk or single-cell sequencing approaches.

Dissecting the Roles of the Cytokinin Signaling Network: The Case of De Novo Shoot Apical Meristem Formation.

Numerous biotechnological applications require a fast and efficient clonal propagation of whole plants under controlled laboratory conditions. For most plant species, the de novo regeneration of shoots from the cuttings of various plant organs can be obtained on nutrient media supplemented with plant hormones, auxin and cytokinin. While auxin is needed during the early stages of the process that include the establishment of pluripotent primordia and the subsequent acquisition of organogenic competence, cytokinin-supplemented media are required to induce these primordia to differentiate into developing shoots. The perception of cytokinin through the receptor ARABIDOPSIS HISTIDINE KINASE4 (AHK4) is crucial for the activation of the two main regulators of the establishment and maintenance of shoot apical meristems (SAMs): SHOOTMERISTEMLESS (STM) and the WUSCHEL-CLAVATA3 (WUS-CLV3) regulatory circuit. In this review, we summarize the current knowledge of the roles of the cytokinin signaling cascade in the perception and transduction of signals that are crucial for the de novo establishment of SAMs and lead to the desired biotechnological output-adventitious shoot multiplication. We highlight the functional differences between individual members of the multigene families involved in cytokinin signal transduction, and demonstrate how complex genetic regulation can be achieved through functional specialization of individual gene family members.

In planta transformation in wheat: an improved protocol to develop wheat transformants.

BACKGROUND: Lack of efficient transformation protocol continues to be a major bottleneck for successful genome editing or transgenic development in wheat. An in planta transformation method was developed in Indian bread wheat in earlier study (Vasil et al. in Nat Biotechnol 10:667-674, 1992) which was labour-intensive and time-consuming. In the present study, in planta transformation method was improved to make it simple, efficient, less labour-intensive and time-saving. METHODS AND RESULTS: PCR-based screening for generated transformants at T0 stage was introduced in this method. Shoot apical meristem of two days old wheat seedling was inoculated with the routine active culture of Agrobacterium tumefaciens harboring plasmid pCAMBIA1300-Ubi-GFP having gene GFP under the control of Zea mays ubiquitin promoter. PCR analysis at T0 stage confirmed 27 plants to be transgene positive. These 27 plants were only taken to the next generation (T1) and the rest were discarded. At T1 generation 6 plants were analyzed to be PCR positive. Out of them, 4 plants were confirmed to have stable integration of transgene (GFP). Fluorescent microscopy at T1 stage confirmed the 4 Southern hybridization positive plants to be expressing reporter gene GFP. CONCLUSIONS: Screening at T0 stage, reduced the load of plants to be taken to T1 generation and their screening thereof at T1 with no overall loss in transformation efficiency. We successfully transformed wheat genotype HD2894 with 3.33% transformation efficiency using a simple, effective method which was less labour-intensive and less time-consuming. This method may be utilized to develop wheat transgenic as well as genome edited lines for desirable traits.

Unraveling the molecular mechanisms governing axillary meristem initiation in plants.

MAIN CONCLUSION: Axillary meristems (AMs) located in the leaf axils determine the number of shoots or tillers eventually formed, thus contributing significantly to the plant architecture and crop yields. The study of AM initiation is unavoidable and beneficial for crop productivity. Shoot branching is an undoubted determinant of plant architecture and thus greatly impacts crop yield due to the panicle-bearing traits of tillers. The emergence of the AM is essential for the incipient bud formation, and then the bud is dormant or outgrowth immediately to form a branch or tiller. While numerous reviews have focused on plant branching and tillering development networks, fewer specifically address AM initiation and its regulatory mechanisms. This review synthesizes the significant advancements in the genetic and hormonal factors governing AM initiation, with a primary focus on studies conducted in Arabidopsis (Arabidopsis thaliana L.) and rice (Oryza sativa L.). In particular, by elaborating on critical genes like LATERAL SUPPRESSOR (LAS), which specifically regulates AM initiation and the networks in which they are involved, we attempt to unify the cascades through which they are positioned. We concentrate on clarifying the precise mutual regulation between shoot apical meristem (SAM) and AM-related factors. Additionally, we examine challenges in elucidating AM formation mechanisms alongside opportunities provided by emerging omics approaches to identify AM-specific genes. By expanding our comprehension of the genetic and hormonal regulation of AM development, we can develop strategies to optimize crop production and address global food challenges effectively.

The Shoot Apical Meristem: An Evolutionary Molding of Higher Plants.

The shoot apical meristem (SAM) gives rise to the aerial structure of plants by producing lateral organs and other meristems. The SAM is responsible for plant developmental patterns, thus determining plant morphology and, consequently, many agronomic traits such as the number and size of fruits and flowers and kernel yield. Our current understanding of SAM morphology and regulation is based on studies conducted mainly on some angiosperms, including economically important crops such as maize (Zea mays) and rice (Oryza sativa), and the model species Arabidopsis (Arabidopsis thaliana). However, studies in other plant species from the gymnosperms are scant, making difficult comparative analyses that help us understand SAM regulation in diverse plant species. This limitation prevents deciphering the mechanisms by which evolution gave rise to the multiple plant structures within the plant kingdom and determines the conserved mechanisms involved in SAM maintenance and operation. This review aims to integrate and analyze the current knowledge of SAM evolution by combining the morphological and molecular information recently reported from the plant kingdom.

The Function of Florigen in the Vegetative-to-Reproductive Phase Transition in and around the Shoot Apical Meristem.

Plants undergo a series of developmental phases throughout their life-cycle, each characterized by specific processes. Three critical features distinguish these phases: the arrangement of primordia (phyllotaxis), the timing of their differentiation (plastochron) and the characteristics of the lateral organs and axillary meristems. Identifying the unique molecular features of each phase, determining the molecular triggers that cause transitions and understanding the molecular mechanisms underlying these transitions are keys to gleaning a complete understanding of plant development. During the vegetative phase, the shoot apical meristem (SAM) facilitates continuous leaf and stem formation, with leaf development as the hallmark. The transition to the reproductive phase induces significant changes in these processes, driven mainly by the protein FT (FLOWERING LOCUS T) in Arabidopsis and proteins encoded by FT orthologs, which are specified as 'florigen'. These proteins are synthesized in leaves and transported to the SAM, and act as the primary flowering signal, although its impact varies among species. Within the SAM, florigen integrates with other signals, culminating in developmental changes. This review explores the central question of how florigen induces developmental phase transition in the SAM. Future research may combine phase transition studies, potentially revealing the florigen-induced developmental phase transition in the SAM.

Single-cell transcriptomic analysis of pea shoot development and cell-type-specific responses to boron deficiency.

Understanding how nutrient stress impacts plant growth is fundamentally important to the development of approaches to improve crop production under nutrient limitation. Here we applied single-cell RNA sequencing to shoot apices of Pisum sativum grown under boron (B) deficiency. We identified up to 15 cell clusters based on the clustering of gene expression profiles and verified cell identity with cell-type-specific marker gene expression. Different cell types responded differently to B deficiency. Specifically, the expression of photosynthetic genes in mesophyll cells (MCs) was down-regulated by B deficiency, consistent with impaired photosynthetic rate. Furthermore, the down-regulation of stomatal development genes in guard cells, including homologs of MUTE and TOO MANY MOUTHS, correlated with a decrease in stomatal density under B deficiency. We also constructed the developmental trajectory of the shoot apical meristem (SAM) cells and a transcription factor interaction network. The developmental progression of SAM to MC was characterized by up-regulation of genes encoding histones and chromatin assembly and remodeling proteins including homologs of FASCIATA1 (FAS1) and SWITCH DEFECTIVE/SUCROSE NON-FERMENTABLE (SWI/SNF) complex. However, B deficiency suppressed their expression, which helps to explain impaired SAM development under B deficiency. These results represent a major advance over bulk-tissue RNA-seq analysis in which cell-type-specific responses are lost and hence important physiological responses to B deficiency are missed. The reported findings reveal strategies by which plants adapt to B deficiency thus offering breeders a set of specific targets for genetic improvement. The reported approach and resources have potential applications well beyond P. sativum species and could be applied to various legumes to improve their adaptability to multiple nutrient or abiotic stresses.

The transcription factor HSFA7b controls thermomemory at the shoot apical meristem by regulating ethylene biosynthesis and signaling in Arabidopsis.

The shoot apical meristem (SAM) is responsible for overall shoot growth by generating all aboveground structures. Recent research has revealed that the SAM displays an autonomous heat stress (HS) memory of a previous non-lethal HS event. Considering the importance of the SAM for plant growth, it is essential to determine how its thermomemory is mechanistically controlled. Here, we report that HEAT SHOCK TRANSCRIPTION FACTOR A7b (HSFA7b) plays a crucial role in this process in Arabidopsis, as the absence of functional HSFA7b results in the temporal suppression of SAM activity after thermopriming. We found that HSFA7b directly regulates ethylene response at the SAM by binding to the promoter of the key ethylene signaling gene ETHYLENE-INSENSITIVE 3 to establish thermotolerance. Moreover, we demonstrated that HSFA7b regulates the expression of ETHYLENE OVERPRODUCER 1 (ETO1) and ETO1-LIKE 1, both of which encode ethylene biosynthesis repressors, thereby ensuring ethylene homeostasis at the SAM. Taken together, these results reveal a crucial and tissue-specific role for HSFA7b in thermomemory at the Arabidopsis SAM.

Unlocking nature's (sub)cellular symphony: Phase separation in plant meristems.

Plant development is based on the balance of stem cell maintenance and differentiation in the shoot and root meristems. The necessary cell fate decisions are regulated by intricate networks of proteins and biomolecules within plant cells and require robust and dynamic compartmentalization strategies, including liquid-liquid phase separation (LLPS), which allows the formation of membrane-less compartments. This review summarizes the current knowledge about the emerging field of LLPS in plant development, with a particular focus on the shoot and root meristems. LLPS regulates not only floral transition and flowering time while integrating environmental signals in the shoots but also influences auxin signalling and is putatively involved in maintaining the stem cell niche (SCN) in the roots. Therefore, LLPS has the potential to play a crucial role in the plasticity of plant development, necessitating further research for a comprehensive understanding.

Nitrogen-Activated CLV3/ESR-Related 4 (CLE4) Regulates Shoot, Root, and Stolon Growth in Potato.

In potato, high levels of nitrogen (N) can lead to excessive vegetative growth at the expense of tuber development, resulting in lower yield and poor-quality tubers. We found that Solanum tuberosum CLE4 (StCLE4) is expressed most strongly in the roots grown in N-rich media, and it positively regulates potato root growth under N-deficient conditions. We noted that StCLE4 functions as a negative regulator of normal shoot apex development similar to CLV3 in Arabidopsis. Transcriptomic analysis revealed that overexpression of StCLE4 resulted in the repression of the StIT1 gene, a regulator of potato tuber initiation. StCLE4-overexpressing stolons were converted into branches, that were similar to a mild phenotype of the it1 (identity of tuber 1) mutant. We also found that NIN-like proteins, key regulators of nitrate signaling bind to the regulatory sequence of StIT1 in a yeast one-hybrid assay. Taken together, our findings suggest that StCLE4 regulates shoot, root, and stolon growth in potato.

Regulation of Flowering Time and Other Developmental Plasticities by 3' Splicing Factor-Mediated Alternative Splicing in Arabidopsis thaliana.

Plants, as sessile organisms, show a high degree of plasticity in their growth and development and have various strategies to cope with these alterations under continuously changing environments and unfavorable stress conditions. In particular, the floral transition from the vegetative and reproductive phases in the shoot apical meristem (SAM) is one of the most important developmental changes in plants. In addition, meristem regions, such as the SAM and root apical meristem (RAM), which continually generate new lateral organs throughout the plant life cycle, are important sites for developmental plasticity. Recent findings have shown that the prevailing type of alternative splicing (AS) in plants is intron retention (IR) unlike in animals; thus, AS is an important regulatory mechanism conferring plasticity for plant growth and development under various environmental conditions. Although eukaryotes exhibit some similarities in the composition and dynamics of their splicing machinery, plants have differences in the 3' splicing characteristics governing AS. Here, we summarize recent findings on the roles of 3' splicing factors and their interacting partners in regulating the flowering time and other developmental plasticities in Arabidopsis thaliana.