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.

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.

Topological properties accurately predict cell division events and organization of shoot apical meristem in Arabidopsis thaliana.

Cell division and the resulting changes to the cell organization affect the shape and functionality of all tissues. Thus, understanding the determinants of the tissue-wide changes imposed by cell division is a key question in developmental biology. Here, we use a network representation of live cell imaging data from shoot apical meristems (SAMs) in Arabidopsis thaliana to predict cell division events and their consequences at the tissue level. We show that a support vector machine classifier based on the SAM network properties is predictive of cell division events, with test accuracy of 76%, which matches that based on cell size alone. Furthermore, we demonstrate that the combination of topological and biological properties, including cell size, perimeter, distance and shared cell wall between cells, can further boost the prediction accuracy of resulting changes in topology triggered by cell division. Using our classifiers, we demonstrate the importance of microtubule-mediated cell-to-cell growth coordination in influencing tissue-level topology. Together, the results from our network-based analysis demonstrate a feedback mechanism between tissue topology and cell division in A. thaliana SAMs.

TPLATE complex-dependent endocytosis attenuates CLAVATA1 signaling for shoot apical meristem maintenance.

Endocytosis regulates the turnover of cell surface localized receptors, which are crucial for plants to rapidly respond to stimuli. The evolutionary ancient TPLATE complex (TPC) plays an essential role in endocytosis in Arabidopsis plants. Knockout or knockdown of single TPC subunits causes male sterility and seedling lethality phenotypes, complicating analysis of the roles of TPC during plant development. Partially functional alleles of TPC subunits however only cause mild developmental deviations. Here, we took advantage of the partially functional TPLATE allele, WDXM2, to investigate a role for TPC-dependent endocytosis in receptor-mediated signaling. We discovered that reduced TPC-dependent endocytosis confers a hypersensitivity to very low doses of CLAVATA3 peptide signaling. This hypersensitivity correlated with the abundance of the CLAVATA3 receptor protein kinase CLAVATA1 at the plasma membrane. Genetic and biochemical analysis as well as live-cell imaging revealed that TPC-dependent regulation of CLAVATA3-dependent internalization of CLAVATA1 from the plasma membrane is required for shoot stem cell homeostasis. Our findings provide evidence that TPC-mediated endocytosis and degradation of CLAVATA1 is a mechanism to dampen CLAVATA3-mediated signaling during plant development.

Rice florigens control a common set of genes at the shoot apical meristem including the F-BOX BROADER TILLER ANGLE 1 that regulates tiller angle and spikelet development.

Rice flowering is triggered by transcriptional reprogramming at the shoot apical meristem (SAM) mediated by florigenic proteins produced in leaves in response to changes in photoperiod. Florigens are more rapidly expressed under short days (SDs) compared to long days (LDs) and include the HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1) phosphatidylethanolamine binding proteins. Hd3a and RFT1 are largely redundant at converting the SAM into an inflorescence, but whether they activate the same target genes and convey all photoperiodic information that modifies gene expression at the SAM is currently unclear. We uncoupled the contribution of Hd3a and RFT1 to transcriptome reprogramming at the SAM by RNA sequencing of dexamethasone-inducible over-expressors of single florigens and wild-type plants exposed to photoperiodic induction. Fifteen highly differentially expressed genes common to Hd3a, RFT1, and SDs were retrieved, 10 of which still uncharacterized. Detailed functional studies on some candidates revealed a role for LOC_Os04g13150 in determining tiller angle and spikelet development and the gene was renamed BROADER TILLER ANGLE 1 (BRT1). We identified a core set of genes controlled by florigen-mediated photoperiodic induction and defined the function of a novel florigen target controlling tiller angle and spikelet development.

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.

ERECTA family signaling constrains CLAVATA3 and WUSCHEL to the center of the shoot apical meristem.

The shoot apical meristem (SAM) is a reservoir of stem cells that gives rise to all post-embryonic above-ground plant organs. The size of the SAM remains stable over time owing to a precise balance of stem cell replenishment versus cell incorporation into organ primordia. The WUSCHEL (WUS)/CLAVATA (CLV) negative feedback loop is central to SAM size regulation. Its correct function depends on accurate spatial expression of WUS and CLV3 A signaling pathway, consisting of ERECTA family (ERf) receptors and EPIDERMAL PATTERNING FACTOR LIKE (EPFL) ligands, restricts SAM width and promotes leaf initiation. Although ERf receptors are expressed throughout the SAM, EPFL ligands are expressed in its periphery. Our genetic analysis of Arabidopsis demonstrated that ERfs and CLV3 synergistically regulate the size of the SAM, and wus is epistatic to ERf genes. Furthermore, activation of ERf signaling with exogenous EPFLs resulted in a rapid decrease of CLV3 and WUS expression. ERf-EPFL signaling inhibits expression of WUS and CLV3 in the periphery of the SAM, confining them to the center. These findings establish the molecular mechanism for stem cell positioning along the radial axis.

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.

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 (RAM) and the shoot apical meristem (SAM). In these meristematic tissues, cells with relatively slower mitotic activity are present in the quiescent center (QC) and the central zone (CZ), respectively. These centers form long-term reservoirs of stem cells maintaining the meristematic stem cell niche (SCN), and thus sustaining continuous plant development and adaptation to changing environments. This review explores the early observations, structural characteristics, functions, and gene regulatory networks of the RAM and SAM. It also highlights the intricate mechanism of dormancy within the SAM. The aim is to contribute to a holistic understanding of quiescence in plants, which is fundamental for the plant proper growth and environmental response.

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.

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.

Genome-wide identification and characterization of the lettuce GASA family in response to abiotic stresses.

BACKGROUND: Lettuce is one of the most extensively farmed vegetables in the world, and it prefers cool growing conditions. High temperatures promote premature bolt formation, reducing quality and yield. The gibberellic acid-stimulated Arabidopsis (GASA) family genes play critical roles in plant growth, development, and stress responses. However, the biological functions of GASA proteins in lettuce have yet to be thoroughly investigated. RESULTS: Using genome-wide analysis, 20 GASAs were identified in lettuce including, three groups of LsGASA proteins based on the phylogenetic analysis. Except for one, all GASA proteins included a conserved GASA domain with 12 cysteine residues. Cis-element analysis showed that LsGASAs were closely associated with light, phytohormones, and stress resistance. Five segmental and three tandem duplication events were observed in the LsGASA family based on duplication analysis. GASA synteny analysis among lettuce, Arabidopsis, tobacco, and rice revealed that LsGASA5 is highly collinear with all species. Six of the 20 LsGASA showed increased expression patterns at specific time points in the shoot apical meristem when subjected to heat stress. According to gene expression analysis, the majority of GASA were highly expressed in flowers compared to other organs, and six GASA exhibited highly increased expression levels in response to NaCl, abscisic acid, and gibberellin treatment. Furthermore, LsGASA proteins are predominantly found in the plasma membrane and/or the cytosol. CONCLUSIONS: This study provides a comprehensive characterization of LsGASA genes for their diversity and biological functions. Moreover, our results will be useful for further studies on the function of lettuce GASA in abiotic stress- and heat-induced bolting signaling.

Circular RNAs modulate the floral fate acquisition in soybean shoot apical meristem.

BACKGROUND: Soybean (Glycine max), a major oilseed and protein source, requires a short-day photoperiod for floral induction. Though key transcription factors controlling flowering have been identified, the role of the non-coding genome is limited. Circular RNAs (circRNAs) recently emerged as a novel class of RNAs with critical regulatory functions. However, a study on circRNAs during the floral transition of a crop plant is lacking. We investigated the expression and potential function of circRNAs in floral fate acquisition by soybean shoot apical meristem in response to short-day treatment. RESULTS: Using deep sequencing and in-silico analysis, we denoted 384 circRNAs, with 129 exhibiting short-day treatment-specific expression patterns. We also identified 38 circRNAs with predicted binding sites for miRNAs that could affect the expression of diverse downstream genes through the circRNA-miRNA-mRNA network. Notably, four different circRNAs with potential binding sites for an important microRNA module regulating developmental phase transition in plants, miR156 and miR172, were identified. We also identified circRNAs arising from hormonal signaling pathway genes, especially abscisic acid, and auxin, suggesting an intricate network leading to floral transition. CONCLUSIONS: This study highlights the gene regulatory complexity during the vegetative to reproductive transition and paves the way to unlock floral transition in a crop plant.

Plant stem-cell organization and differentiation at single-cell resolution.

Plants maintain populations of pluripotent stem cells in shoot apical meristems (SAMs), which continuously produce new aboveground organs. We used single-cell RNA sequencing (scRNA-seq) to achieve an unbiased characterization of the transcriptional landscape of the maize shoot stem-cell niche and its differentiating cellular descendants. Stem cells housed in the SAM tip are engaged in genome integrity maintenance and exhibit a low rate of cell division, consistent with their contributions to germline and somatic cell fates. Surprisingly, we find no evidence for a canonical stem-cell organizing center subtending these cells. In addition, trajectory inference was used to trace the gene expression changes that accompany cell differentiation, revealing that ectopic expression of KNOTTED1 (KN1) accelerates cell differentiation and promotes development of the sheathing maize leaf base. These single-cell transcriptomic analyses of the shoot apex yield insight into the processes of stem-cell function and cell-fate acquisition in the maize seedling and provide a valuable scaffold on which to better dissect the genetic control of plant shoot morphogenesis at the cellular level.

Salinity-Triggered Responses in Plant Apical Meristems for Developmental Plasticity.

Salt stress severely affects plant growth and development. The plant growth and development of a sessile organism are continuously regulated and reformed in response to surrounding environmental stress stimuli, including salinity. In plants, postembryonic development is derived mainly from primary apical meristems of shoots and roots. Therefore, to understand plant tolerance and adaptation under salt stress conditions, it is essential to determine the stress response mechanisms related to growth and development based on the primary apical meristems. This paper reports that the biological roles of microRNAs, redox status, reactive oxygen species (ROS), nitric oxide (NO), and phytohormones, such as auxin and cytokinin, are important for salt tolerance, and are associated with growth and development in apical meristems. Moreover, the mutual relationship between the salt stress response and signaling associated with stem cell homeostasis in meristems is also considered.

Efficient genotype-independent cotton genetic transformation and genome editing.

Cotton (Gossypium spp.) is one of the most important fiber crops worldwide. In the last two decades, transgenesis and genome editing have played important roles in cotton improvement. However, genotype dependence is one of the key bottlenecks in generating transgenic and gene-edited cotton plants through either particle bombardment or Agrobacterium-mediated transformation. Here, we developed a shoot apical meristem (SAM) cell-mediated transformation system (SAMT) that allowed the transformation of recalcitrant cotton genotypes including widely grown upland cotton (Gossypium hirsutum), Sea island cotton (Gossypium barbadense), and Asiatic cotton (Gossypium arboreum). Through SAMT, we successfully introduced two foreign genes, GFP and RUBY, into SAM cells of some recalcitrant cotton genotypes. Within 2-3 months, transgenic adventitious shoots generated from the axillary meristem zone could be recovered and grown into whole cotton plants. The GFP fluorescent signal and betalain accumulation could be observed in various tissues in GFP- and RUBY-positive plants, as well as in their progenies, indicating that the transgenes were stably integrated into the genome and transmitted to the next generation. Furthermore, using SAMT, we successfully generated edited cotton plants with inheritable targeted mutagenesis in the GhPGF and GhRCD1 genes through CRISPR/Cas9-mediated genome editing. In summary, the established SAMT transformation system here in this study bypasses the embryogenesis process during tissue culture in a conventional transformation procedure and significantly accelerates the generation of transgenic and gene-edited plants for genetic improvement of recalcitrant cotton varieties.

Plant gene silencing signals move from the phloem to influence gene expression in shoot apical meristems.

BACKGROUND: Small RNAs (sRNA) are potent regulators of gene expression that can diffuse short distances between cells and move long distances through plant vasculature. However, the degree to which sRNA silencing signals can move from the phloem to the shoot apical meristem (SAM) remains unclear. RESULTS: Two independent transgenic approaches were used to examine whether phloem sRNA silencing can reach different domains of the SAM and silence SAM-expressed genes. First, the phloem companion-cell specific SUCROSE-PROTON SYMPORTER2 (SUC2) promoter was used to drive expression of an inverted repeat to target the FD gene, an exclusively SAM-localized floral regulator. Second, the SUC2 promoter was used to express an artificial microRNA (aMiR) designed to target a synthetic CLAVATA3 (CLV3) transgene in SAM stem cells. Both phloem silencing signals phenocopied the loss of function of their targets and altered target gene expression suggesting that a phloem-to-SAM silencing communication axis exists, connecting distal regions of the plant to SAM stem cells. CONCLUSIONS: Demonstration of phloem-to-SAM silencing reveals a regulatory link between somatic sRNA expressed in distal regions of the plant and the growing shoot. Since the SAM stem cells ultimately produce the gametes, we discuss the intriguing possibility that phloem-to-SAM sRNA trafficking could allow transient somatic sRNA expression to manifest stable, transgenerational epigenetic changes.

MicroRNA172 controls inflorescence meristem size through regulation of APETALA2 in Arabidopsis.

The APETALA2 (AP2) transcription factor regulates flower development, floral transition and shoot apical meristem (SAM) maintenance in Arabidopsis. AP2 is also regulated at the post-transcriptional level by microRNA172 (miR172), but the contribution of this to SAM maintenance is poorly understood. We generated transgenic plants carrying a form of AP2 that is resistant to miR172 (rAP2) or carrying a wild-type AP2 susceptible to miR172. Phenotypic and genetic analyses were performed on these lines and mir172 mutants to study the role of AP2 regulation by miR172 on meristem size and the rate of flower production. We found that rAP2 enlarges the inflorescence meristem by increasing cell size and cell number. Misexpression of rAP2 from heterologous promoters showed that AP2 acts in the central zone (CZ) and organizing center (OC) to increase SAM size. Furthermore, we found that AP2 is negatively regulated by AUXIN RESPONSE FACTOR 3 (ARF3). However, genetic analyses indicated that ARF3 also influences SAM size and flower production rate independently of AP2. The study identifies miR172/AP2 as a regulatory module controlling inflorescence meristem size and suggests that transcriptional regulation of AP2 by ARF3 fine-tunes SAM size determination.

Role for the shoot apical meristem in the specification of juvenile leaf identity in Arabidopsis.

The extent to which the shoot apical meristem (SAM) controls developmental decisions, rather than interpreting them, is a longstanding issue in plant development. Previous work suggests that vegetative phase change is regulated by signals intrinsic and extrinsic to the SAM, but the relative importance of these signals for this process is unknown. We investigated this question by examining the effect of meristem-deficient mutations on vegetative phase change and on the expression of key regulators of this process, miR156 and its targets, SPL transcription factors. We found that the precocious phenotypes of meristem-deficient mutants are a consequence of reduced miR156 accumulation. Tissue-specific manipulation of miR156 levels revealed that the SAM functions as an essential pool of miR156 early in shoot development, but that its effect on leaf identity declines with age. We also found that SPL genes control meristem size by repressing WUSCHEL expression via a novel genetic pathway.

Identification and Fine Mapping of the Candidate Gene Controlling Multi-Inflorescence in Brassica napus.

As a desirable agricultural trait, multi-inflorescence (MI) fulfills the requirement of mechanized harvesting and yield increase in rapeseed (Brassica napus L.). However, the genetic mechanism underlying the multi-inflorescence trait remain poorly understood. We previously identified a difference of one pair of dominant genes between the two mapping parental materials. In this study, phenotype and expression analysis indicated that the imbalance of the CLAVATA (CLV)-WUSCHEL (WUS) feedback loop may contribute to the abnormal development of the shoot apical meristem (SAM). BnaMI was fine-mapped to a 55 kb genomic region combining with genotype and phenotype of 5768 BCF1 individuals using a traditional mapping approach. Through comparative and expression analyses, combined with the annotation in Arabidopsis, five genes in this interval were identified as candidate genes. The present findings may provide assistance in functional analysis of the mechanism associated with multi-inflorescence and yield increase in rapeseed.