The Shoot Apical Meristem: An Evolutionary Molding of Higher Plants

1. Introduction

Plants can maintain indeterminate postembryonic growth by reserving pools of stem cells. These reservoirs are confined to specialized regions called meristems, which are present in various plant structures. The apical meristems (AMs), located on the tips of the germinating seedling, are responsible for the plant's primary growth or length. There are two types of AMs: the shoot apical meristem (SAM) and the root apical meristem (RAM), which are responsible for the growth of the aerial part and the root system of plants, respectively. Additionally, plants have lateral meristems, which are responsible for secondary growth that determines the thickness of the plant, and intercalary meristems, which are responsible for growth at the base of the nodes and leaf blades in monocots [1].

Beyond growth, meristems also play a crucial role in defining plant anatomical patterns. With noticeable differences in their structure, meristems serve as a valuable model for studying plant evolution and understanding the structural and functional aspects of plant development. In general, primitive plants have a simple meristem consisting of only one apical cell, whereas late divergent plants have multicellular meristems [2]. Despite these important structural variations, meristematic cells share two essential and highly conserved functions: maintaining the meristem integrity, including its stem cell population, and producing cells to support plant growth [3]. This review summarizes current knowledge on meristem structure, function, and evolution. Given the broad scope of this topic and the scarcity of studies dedicated exclusively to studying the SAM from an evolutionary perspective, we integrate and synthesize all relevant literature on meristem reserach. The goal is to provide a comprehensive review that facilitates the understanding of SAM evolution. We divide this review into four main sections:

• In the first section, we present essential concepts to understand plant meristems. We introduce the concept of stem cells and compare the stem cell niche of RAM and SAM. We emphasize the importance of plant life cycles, as this concept gives rise to SAM evolution theories.
• Then, the meristem shape is analyzed throughout phylogeny. This section offers a morphological description of SAM across plant evolution. Given the extensive literature on SAM morphology, it provides perspectives on the differences in SAM between clades.
• We provide a comprehensive review of the regulatory and maintenance mechanisms in the SAM. This section focuses on the regulatory loops described for angiosperms and the conserved elements across clades.
• Taking advantage of single-cell transcriptomics to understand SAM, this section delves into the research of single-cell transcriptomics and single-nucleus transcriptomics on SAM. We describe key studies and discuss their findings.

2. Evolutionary Origin of the Meristem

2.1. The Concept of Stem Cells

Plant stem cells are innately undifferentiated and are contained mainly in the AMs. They are commonly defined by their developmental capabilities, namely, self-renewal and the ability to differentiate into multiple cell types [4]. Therefore, one of the main interests in studying AMs is related to their stem cell pool. Plant cells can be de-differentiated and re-differentiated into various cell types, which is why they are considered totipotent. The term totipotency refers to the capacity of one cell to produce different tissues and eventually regenerate into an entire organism [5]. However, the specific conditions that activate their differentiation into different cell types are still largely unknown [6]. To study this process, the standard models in plants are embryos [7], protoplasts [8], calli [9], and meristems [10]. These models are valuable as they contain dedifferentiated cells, which can develop into differentiated cells, ultimately giving rise to different plant tissues. Hence, an interesting perspective to understand this phenomenon is to focus on the evolution of totipotency by comparing the meristems of early and modern diverged land plants, signaling out the common factors on totipotency through evolution.

2.2. SAM and RAM

Extensive research has been conducted to understand RAM evolution, but little is known about the SAM evolutionary process. Since both AMs are structures with similar characteristics, studying the RAM can be a starting point for understanding the SAM. Both are reservoirs of self-renewing stem cells that can be considered primary meristems because, in most cases, they are determined during embryogenesis [11]. Stem cells in the RAM and SAM are located in regions referred to as the quiescent center (QC) and organizing center (OC), respectively. The QC and the OC are the signaling centers responsible for stem cell maintenance in both meristems [12] and have been proposed to be functionally equivalent [13,14].

Studies conducted on the model plant Arabidopsis thaliana suggest that the organization of the signaling components required for stem cell initiation and maintenance in the RAM and SAM are relatively conserved. Consequently, similar classes of genes have been co-opted as central regulators in both types of meristems [14,15]. However, despite these shared mechanisms, the hormonal function has been shown to be inverse [16,17]. In the SAM, auxins trigger differentiation, whereas in the RAM, they maintain the stem cell niche and support cell proliferation [18]. On the other hand, in the SAM, cytokinin promotes tissue proliferation of undifferentiated cells, whereas it promotes cell differentiation in the RAM [19,20]. The OC of the SAM is the site of maximal cytokinin activity, whereas the auxin maximum is in the QC of the RAM [20] (Figure 1).

Fossil evidence suggests that the RAM evolved at least twice independently rather than having a single origin [21,22]. The first appearance occurred in the lycophytes clade, followed by a second evolutionary event, which likely occurred in the ancestor of euphyllophytes (vascular plants non-lycophytes) [23]. It has even been proposed that RAM independently evolved multiple times in lycophytes [24]. Moreover, the similarities between lycophyte and euphyllophyte roots, including indeterminate growth, apical meristem protected by a root cap, root hairs, and a stele covered by a specialized endodermal cell layer, are clear examples of convergent evolution [25,26]. Genomic analyses revealed limited gene expansion occurred at the divergence between the lycophyte and euphyllophyte clades [27]. Consequently, all the similarities produced by convergent evolution did not require extra gene families, suggesting that the rewiring of existing genetic programs was sufficient to generate multiple independent emergences of the RAM [28].

2.3. Apical Metistem of Gametophyte and Sporophyte

The plant life cycle comprises two alternating phases: the haploid or gametophyte phase and the diploid or sporophyte phase. The diploid phase concludes with meiosis, while the haploid phase ends with the generation and union of gametes [31]. It is remarkable that, as land plants diversified, the sporophyte generation became increasingly specialized, whereas the gametophyte generation became reduced [32,33]; This tendency is reflected in SAMs, where non-seed plants usually have single-celled SAMs to sustain the growth of the sporophytes and gametophytes. In contrast, seed plants' sporophytes develop complex multicellular meristems, while their gametophytes lack meristems [34,35] (Figure 2). However, as both phases have SAMs, it is reasonable to discuss the origin of meristems in terms of the plant life cycle.

Two hypotheses have been proposed that explain the appearance of the alternation cycle [36]. First, the homologous theory describes the ancestor of land plants with an originally haplontic life cycle that gives rise to a sporophyte through delay in meiosis, resulting in the intercalation of a new diploid organism [33,37]. This assumption outlines the gametophytic SAM as the ancestor of the sporophytic SAM. The second hypothesis is the antithetic theory, which proposes that sporophytic SAM is independent of gametophytic SAM. According to this theory, the sporophytic SAM evolved de novo by intercalating a system between embryonic and reproductive growth [33,38]. Current evidence based on embryophytes and fossil records is more compatible with the antithetic theory than the homologous theory [39,40]. Hypotheses derived from the antithetic theory include 1) the embryophyte SAM originated from the transient seta meristem (sporophyte) of a bryophyte, 2) the hornwort sporophyte meristem is the possible ancestor of the SAM, and 3) the SAM in vascular plants arose de novo from a bryophyte ancestor [41].

Overall, evidence suggests that gametophyte and sporophyte SAM have an independent origin. However, as plants share the same startup gene toolkit since their separation from algae, they probably also share homologous regulatory loops and mechanisms on their SAM regulation. In fact, mosses and ferns gametophyte SAMs share key transcription factors (TFs) with angiosperm sporophyte SAMs [42,43], which suggests the existence fundamental mechanisms involved in stem cell regulation across land plants.

3. Meristem Shape Throughout Phylogeny

A traditional approach to studying meristems considers their morphology and focuses on their correlation to plant growth patterns. Cell division patterns and apical cell symmetry can outline plant morphology [44]. For example, in bryophytes, taxa with tetrahedrical apical cells tend to have leafy morphologies, while taxa with hemidiscoid apical cells tend to be thalloid [45]. The study of apical cells applies mainly to SAMs with a single apical cell found in macroalgae, bryophytes, lycophytes, and ferns. The simplest apical cell is the dome-shaped cell present in macroalgae (Charales) and moss gametophytes, and it allows one-dimensional (1D) growth. However, land plant clades such as lycophytes and ferns present a tetrahedral apical cell allowing three-dimensional (3D) growth [46,47] (Figure 3). It can be hypothesized that the emergence of apical cells capable of dividing following multiple planes resulted in drastic morphological innovations, ultimately allowing the colonization of land by plants.

3.1. Algae

The evolution from algae to plants is closely tied to the evolution of the body plan of plants. The Charophycean green algae, the closest relative of the extant land plants [50,51], provides an evolutionary example of the transition from unicellular to multicellularity. Some Chlorophycean algae are still at the midpoint of this transition [52]. However, the presence of a SAM is limited to multicellular organisms with organized tissues.

Among Charophyceans, apical growth has been demonstrated only in the order Charales, which have a single apical cell per shoot [53]. The dome-shaped apical cells of Charales have one cutting face opposite the apex of the surface, and this cutting plane is parallel to the orientation of the last cell plate. As a result, they present 1D growth, forming cell chains resembling filaments or mats [46,54]. Another charophyte, the Coleochaetalean algae, typically exhibits a marginal meristem where cells can divide into two possible planes, leading to the formation of radially symmetrical filamentous or monochromatic thalli that resemble hornworts gametophytes [55].

3.2. Bryophytes

Bryophytes comprise a taxonomic group consisting of three clades of non-vascular land plants: mosses, liverworts, and hornworts [56]. It is traditionally considered that land plant traits primarily evolved in bryophytes. Liverworts were the earliest-divergent clade of land plants, whereas mosses and hornworts are sister groups. However, there is only partial acceptance of the internal topology of this clade, despite evidence of its monophyly [57,58]. It has been proposed that the sporophyte SAM was derived from a sterilized bryophyte sporangium, but this theory remains untestable due to a lack of homologies or fossil records [59,60].

The moss sporophyte has three transient cells recognized as meristems: the apical cell, active in the early embryo; the central seta cell that maintains the seta's growth; and a transient single apical cell of the sporophytic tissue [61]. This anatomy is similar to that of liverworts, except that, in mosses, the seta arises from a transient meristem developing in the middle of the spindle-shaped embryo. In contrast, hornworts lack a seta and, consequently, a seta meristem on their sporophyte. The hornwort sporophyte is composed of a foot and a sporangial axis, growing from a basal meristem that remains active throughout the sporophyte's life [33,62]; they possess a unifacial and multicellular basal meristem, called the basal meristem, which provides upwards indeterminate growth. This type of "indeterminate" growth is unprecedented to any fossil or living plant and is similar to the indeterminate growth of sporophytes in tracheophytes. However, the resemblance to the angiosperm SAM is considered unrelated or superficial [63,64]. The hornwort's indeterminate growth contrasts with the limited proliferative growth of sporophytes in mosses and liverworts.

Bryophytes have an extended gametophyte phase during their life cycle. The gametophyte of bryophytes presents a long-lasting single-celled SAM composed of an apical cell (AC). Among bryophytes, mosses exhibit a singular development compared to hornworts and liverworts [34]. After spore germination, mosses develop filamentous tissues called protonema, consisting of two substages. In the early stage of germination, the protonema is composed of chloronemata filaments. The chloronemata exhibits 1D growth during filament extension until an apical stem cell transitions into a caulonemal apical stem cell. The caulonema consists of an extended 2D filamentous stage. Both substages comprise a protonema apical cell. Lastly, moss initiates 3D growth and leafy shoot buds called gametophores with a single tetrahedral apical cell [54]. In contrast, hornworts and liverworts do not possess a protonemal stage; after spore germination, they grow directly into thalli or "leafy" forms [48,65]. Liverworts grow from apical cells located in an invaginated notch. There are four different types of apical cells found in this group. These include a tetrahedral cell with three cutting faces, a Wedge-shaped with two lateral, one dorsal, and one ventral cutting faces, a lenticular or lens-shaped cell with two lateral cutting faces, and a hemidiscoid cell with two lateral and one posterior cutting face [48]. On the other hand, hornwort gametophytes usually present wedge-shaped apical cells with four cutting faces located in notches around the thallus [66].

3.4. Tracheophytes

Numerous efforts have been made to categorize the SAMs of tracheophytes, primarily through their histological and cytological features. It is generally accepted that there are three types of SAMs. This classification is based on the division plane within the SAM cells and the location and number of initial cells [67,68]. Initial cells divide to maintain the meristem as a continuing source of new cells. The three types of tracheophyte SAMs are 1) monoplex type, usually capped by one apical initial cell, also known as apical cell, that contributes to stem growth [69]. This SAM type is found on some lycophytes and ferns; 2) simplex type, whose configuration contains multiple initial cells within a single zone. These are common in gymnosperms [69]; and 3) duplex meristem type that incorporates initial cells distributed across at least two layers [69,70]. These are prevalent in angiosperms.

The indeterminate meristem of tracheophytes is a crucial innovation in plant evolution, which pushed the sporophyte life cycle as the dominant phase in the plant's life cycle [71]. Lycophytes comprise a taxonomic group representing a small portion of vegetal diversity. This clade consists of three living orders: Lycopodiales, Isoëtales, and Selaginellales. However, there are many other extinct orders [72]. Lycophytes display considerable variation in the organization of their meristems, ranging from the monoplex meristem with one or two apical cells found in Selaginellales [73,74] to the simplex meristems found in Lycopodiales and Isoëtales [70]. Monilophytes (ferns) usually have a meristem composed of an apical initial cell and its surrounding cells, this multicellular structure has also been proposed for Selaginella [42,75]. Apical cells of Lycophytes and Monilophytes (ferns) are considered convergent structures [42,76].

SAMs in seed plants tend to be larger and more complex compared to other land plants. They consist of multiple initial cells and distinct functional layers. The outer layer (L1), also known as tunica, gives rise to the epidermis; and L2 in maize, or L2 and L3 (corpus) in Arabidopsis are the internal layers [77,78,79]. It has been suggested that each layer comprises its own group of initials [77]. Gymnosperms SAMs lack tunica but its cells in the upper portion of the gymnosperm SAM form a lens-shaped zone with cytological features similar to those of angiosperms. Remarkably, there are gymnosperm genera with an apparent or discrete outer L1 layer, such as Gnetum and Ephedra [70].

Irrespective of cell layers, in both gymnosperms and angiosperms SAMs exist zonation patterns (known as well as cellular domains), that mirror differences in cell division or cellular activity. Different zones of the meristem possess different functions. Typically, there is a central zone (CZ) surrounded by a peripheral zone (PZ) on the flanks and a rib zone (RZ) behind the CZ. Cells within the CZ self-renew and replenish the surrounding (PZ) and subtending (RZ). Cells within the PZ and RZ proliferate more rapidly than in the CZ and ultimately differentiate to produce lateral organs and ground tissue, respectively [80,81].

4. Shoot Apical Meristem Regulation and Maintenance

The phenomena of branch development along the primary axis of plants, known as apical dominance, have been studied since the 1930s [82,83,84]. Over the course of nine decades of research, a general concept emerged: the phytohormone auxin produced by the shoot tip is transported in a basipetal fashion by the polar transport stream, which inhibits axillary bud outgrowth [82,85]. Although this concept has been observed in the vegetative developmental stages of many short-lived plants and the juvenile stages of some trees [86], it has not been validated in perennial plants [87,88] and remains obscure in plants with dichotomous branching.

Auxin was the first plant regulator explored in SAM activity, and much has been learned about its signaling pathways in different organs. However, particular attention has been given to its interaction with cytokinin, another plant hormone. Auxins and cytokinins act synergistically or antagonistically to control SAM organization, formation, and maintenance [89]. For example, in Arabidopsis, cytokinins promote cell expansion, increasing SAM size, while auxin indirectly promotes differentiation through multiple mechanisms [90]. Despite being the most extensively studied regulators of plant development across plants, our understanding of the crosstalk between these hormones is still limited to a few plant models.

4.1. The Regulatory Model: Angiosperms

With advances in molecular biology, SAM research has expanded from physiological aspects to the regulatory molecular mechanism. Currently, we understand that phytohormones and TFs cooperate to balance meristem maintenance and organ production. The canonical model of SAM regulation is based on the plant model Arabidopsis thaliana, and stands around the key genes CLAVATA (CLV, encoding ligand peptides) and WUSCHEL (WUS, encoding a homeobox TF) [91]. This regulatory loop also involves SHOOT MERISTEMLESS (STM), a KNOTTED1-LIKE HOMEOBOX (KNOX) TF [10,92]. The CLV gene family comprises the CLV peptides CLV3, CLV2, and CLV1. In this regulatory loop, WUS promotes stem cell identity, and is regulated by CVL and STM. In addition, CLV favors organ initiation, and STM prevents the incorporation of central meristem cells into organ primordia [91,93] (Figure 4).

Other genes, such as the plant-specific GRAS TFs, and HAIRY MERISTEM (HAM1–HAM4), interact with WUS/WOX5 (another member of the WUS family) [94]. A HAM concentration gradient modulates the WUS-CLVs interaction, promoting zonation of the SAM. In addition, members of the NAC group of leucine-rich TFs, including CUP-SHAPED COTYLEDON1 (CUC1), CUC2, and CUC3 [95], repeat receptor-like kinase genes (LRR-RLK), REVOLUTA (REV, a homeodomain TF), and the Apetala 2 (AP2) TF family, have been shown to have critical functions in the SAM [96,97].

4.2. SAM TFs Conserved Throughout Evolution

KNOX TFs

The KNOX genes belong to a large family of TFs called homeobox. They are involved in morphogenesis in all major eukaryotic lineages. In plants, members such as KN1, STM, and KNAT are directly associated with SAM maintenance [53]. Conservation of the KNOX gene family can be traced back to the plant's ancestors, including Chlorophytes and Charophycean algae [53]. It has been suggested that KNOX transcription factors are part of the genetic toolkit that enabled the development of multicellularity, cell differentiation, and increased SAM complexity in seed plants [98]. Their functional roles are diverse across the phylogeny, including activation of the diploid phase, sporophyte and spore formation, meristem maintenance, and organogenesis [99,100]. In organisms such as Chlamydomonas reinhardtii, KNOX, and BELL TFs are inherited by gametes of the opposite mating types and heterodimerize in zygotes to activate diploid development [100,101]. Similarly, in Marchantia polymorpha, the expression in the gametophyte of KNOX and BELL is required to initiate zygotic development, while in Physcomitrella patens, KNOX expression is associated with sporophyte development and meristem regulation rather than the gametophyte [62]. In Lycophytes such as Sellaginella and spermatophytes, KNOX genes have been associated with cell proliferation and meristem maintenance [102,103] Unfortunately, until now, there is not a clear pattern of KNOX function across the phylogeny. However, duplication events followed by a sub-functionalization within each lineage appear to explain the presence of paralogs specialized for several developmental functions [53].

MADS TFs

The MADS-box gene family encodes TFs with a DNA-binding MADS domain, which was named after the proteins MINICHROMOSOME MAINTENANCE 1, AGAMOUS, DEFICIENS, and SERUM RESPONSE FACTOR (SRF). They are classified into two major classes based on their structure and phylogeny, Type I and Type II [104]. Type II classic MADS-box genes have been particularly well studied, as many of them have roles in determining floral organs. Type II MADS-box genes further diverged into two groups: MIKCC and MIKC* a [105]. Phylogenetic analyses suggest that algae MIKCC MADS-box genes could be considered as the ancestral MIKCC before their divergence into the MIKC* and MIKCc clades. In Charophycean algae, MIKCC genes play a role in gamete differentiation [106]

In non-seed plants, MIKCC have roles in gametophytic and sporophytic generations and contribute to the development of vegetative and reproductive structures [107,108,109]. However, in seed plants, their function is primarily linked to sporophyte development and the determination of floral organs [110,111]. On the other hand, MIKC* genes of non-seed and seed plants have a conserved role during gametophyte development [109,112]. It has been hypothesized that the function of MADS-box genes became restricted to specific plant organs after duplication events coinciding with the diversification of seed plants. Before that, MADS-box genes had multiple roles throughout plant development [113].

AP2/ERF TFs

The plant-specific APETALA 2/ethylene-responsive factor (AP2/ERF) family is characterized by the AP2 DNA-binding domain [114]. AP2/ERF genes are divided into classes based on the number of AP2 domains present. ERF-like genes contain one AP2 domain, while AP2-like genes contain two AP2 domains [115]. AP2-like genes can be further divided into the clades euANT, basalANT, and euAP2 according to the amino acid sequence of the double AP2 domain and the nuclear localization sequence [116]. In the model plant Arabidopsis thaliana and other angiosperms, all AP2-like clades play key roles in developmental processes and SAM regulation [98,117,118].

The AP2/ERF family is involved in diverse roles through plant evolution. Although there is no experimental evidence of AP2-like function and no ANT sequences in algae. Phylogenetic analysis showed that an AP2 domain (AP2-R1 AA insertion) of the microalgae C. reinhardtii and Chlorokybus atmophyticus form a sister clade to the major clade of the AP2-like sequences of plants. However, they are distinct from euANTgene sequences, forming their own clade [119]. This suggests that C. reinhardtii and C. atmophyticus AP2 sequences could represent the putative ancestral sequence of the ANT group. In contrast, the AP2-like genes of multicellular algae, such as Mesotaenium caldariorum and Klebsormidium nitens, form a clade with land plants. It has been suggested that the ancestor of embryophytes may have a preANT-like gene which gave rise to the land plant exclusive basalANT and euANT lineages [118,120]. However, the diverse functions of this gene family increased following the plant's evolutionary novelties; for example, in ferns, the expression of ANT has been reported in young sporangia, gametes, and spores, in gymnosperms in the ovule and during seed development. In angiosperms, they are involved in the meristem, flower organ, and fruit development [118,121,122].

5. Taking Advantage of Single-Cell Transcriptomics to Understand SAM

Studies on SAM transcriptional pathways have evolved from early techniques, such as in situ hybridization and microdissection to more high-throughput methods, such as single-cell RNA-sequencing (sc-RNA-seq) and single-nucleus RNA-sequencing (sn-RNA-seq). In situ hybridization is used to map specific genes, making it difficult to obtain a comprehensive view of the transcriptome. Microdissection involves dissecting tissue pieces, from which RNA is extracted for analysis. Unfortunately, microdissection studies are susceptible to RNA degradation and low yields, potentially leading to low-quality data [122,123]. scRNA-seq and snRNA-seq (sc/snRNA-seq) is a technique that avoids these problems. sc/snRNA-seq relies on microfluidics (10X Genomics) to capture single cells or nuclei and obtain an RNA sequencing library for each cell or nuclei. After sequencing, the transcriptomes of individual cells are used to group cells into clusters, and clusters are assigned to cell types using previously described markers from fluorescent protein reporters or in situ hybridization [124].

sc/snRNA-seq simultaneously allows the study of cellular dynamics, including the cell cycle, cell differentiation, and gene regulatory network construction [125]. This is particularly useful in studying meristem development, as it allows the identification of stem cells and their exploration during transitional states up to their differentiation [126]. SAM transcriptomic landscapes are a turning point in plant development research. Sc/snRNA-seq offers the possibility to study division and differentiation at cell resolution, quantifying the contribution of individual cells during the formation of new aerial structures, a task that was impossible before this technology [11]. Currently, three SAM scRNA-seq studies in Arabidopsis [127], one in maize [128], and one in pea [129], and two SAM snRNA-seq studies, one in tomato [130] and one in Populus [131], are available.

The transcriptomic landscape of the Arabidopsis SAM is an invaluable reference due to its status as the most extensively studied plant model species and has led to numerous resources for single-cell analysis. Three main conclusions can be drawn from Arabidopsis SAM scRNA-seq research. First, despite the wealth of available markers, there remain undefined groups of cells, hinting at the potential existence of new cell types. Second, this research also explores the dynamics of transcriptomic changes during temporal processes, enabling the assignment of cells into distinct cell cycle phases, which revealed a cell cycle continuum in meristematic tissues, suggesting that subtle variations in cell division duration and patterns play a role in the formation of different cell types. And third, the overlap of single-cell RNA sequencing data from Arabidopsis RAM and SAM uncovered previously unrecognized similarities between shoot and root apex epidermis and vascular tissues, adding to our understanding of plant development [127].

Conde and colleagues [131] developed a SAM cross-species analysis between Populus and Arabidopsis at single-cell resolution. These results provide novel insights into the conservation of transcriptional programs during primary vasculature formation and yield valuable information about xylem and wood formation. The tomato SAM transcriptomic landscape described by Tian and colleagues [130] showed the enrichment of Arabidopsis homologous genes associated with the cell-specific domains, such as meristem, epidermis, mesophyll, and vasculature. Furthermore, Tian and colleagues [130] used a single-cell approach to infer gene regulatory networks, facilitating the identification of key regulators.

For the first time, Chen and colleagues [129] used sc-RNA-seq technology on pea SAMs to understand plant growth under nutrient stress, specifically boron (B) deficiency. Their study links the progression of SAM development with the up regulation of genes encoding histones and chromatin assembly and remodeling proteins under B deficiency conditions. The expression of these genes was suppressed, suggesting a reorganization of chromatin during SAM development and a possibly impaired SAM activity under B deficiency. These results emphasize the importance of considering cell type-specific stress responses, which may be key to unraveling complex biological processes [129].

Satterly and colleagues [128] obtained a transcriptional landscape of the maize SAM. Maize belongs to the monocot clade, while Arabidopsis, Populus, tomato, and pea are eudicots, and one would expect to observe more differences between the transcriptional profiles of monocots vs eudicots. Based on their scRNA-seq data, Satterly and colleagues [128] did not find evidence of a canonical WUS-CLV3 stem-cell OC as in other SAM transcriptional landscapes. The Arabidopsis SAM exhibits a repression mechanism of the stem-cell-promoting transcription factor WUS, which relies on the peptide CLV3 and its receptor CLV1, which surrounds the OC. WUS is crucial in maintaining the stem-cell fate in SAM and floral meristems (FM). However, in maize SAM, there is no clear regulatory loop similar to that in Arabidopsis.

Maize SAM has several Leucine-Rich-Repeat (LRR) receptors and their peptide ligands that could be involved in stem cell control [132,133], but the identity of the TF(s) that promote the stem-cell fate remains unclear. WUS1 has been considered an essential meristem size regulator in maize [96]. Its expression has been detected in the inflorescence meristem (IM) and FM [134]. In addition, an in-situ hybridization localizes WUS in SAM, but against Arabidopsis, it is not restricted to an OC; it is detected in peripheral cell layers at the primordium height [135]. Remarkable in maize, SAM sc-RNA-seq did not detect WUS or any other functionally homologous to WUS [128]. Laureyns and colleagues [136] develop an in-situ sequencing (ISS) on maize SAM meristem. They probed the expression of 90 genes and could not obtain a clear expression pattern for 15 of them, including ZmWUS1 and ZmWUS2.

Several challenges deriving from the protoplasting technique could hinder Maize SAM heterogeneity due to several issues arising from this process. Protoplasting has been linked with issues such as ectopic gene expression, bias on proportions of cell types [8,127], and noise from generation-related gene expression [96,130]. Therefore, it is possible that WUS-containing cells were affected by protoplasting or its low expression level could not capture by the in-situ sequencing (ISS). Considerations on this phenomenon are: first, the expression of WUS in maize meristem varies depending on its developmental state vegetative (SAM), IM, and FM. Second, no evidence exists that maize SAM expresses a canonical OC in normal conditions. Third, WUS functions as a mobile TF [134], so it is possible that we can only see the location of its expression or accumulation. Remarkably, this example in angiosperms between monocots and dicots exposes the possibility of different mechanisms or regulatory mechanisms on their SAM regulation. Therefore, it is exciting to think about the diversity in SAM regulation on different clades.

Using sc/snRNA-seq to study the SAM in non-model plant species has additional challenges. Sc/snRNA-seq relies on previous knowledge of cell type-specific markers to assign cell clusters to cell types. Such markers are rare to non-existent in non-model plant species. Spatial transcriptomics has emerged as an alternative to overcome this limitation, although it has drawbacks. The spatial transcriptomics technique enables creating in situ libraries, thus obtaining transcriptomic landscapes from tissue sections, avoiding the need to cluster and identify each cell population [137]. Giacomello and colleagues [138] established the basis of the spatial transcriptomics method for SAMs of different plant species [11,139]. The possibility of matching the anatomical and molecular information and accelerating gene and tissue discovery is a valuable resource for future functional genomics studies. Additionally, technologies such as spatial transcriptomics and snRNA-seq will facilitate the study of non-model species as it becomes unnecessary to develop protocols for protoplasting or extensive background on marker genes for particular species. These technologies alleviate the technical difficulties of working on SAMs, as seen in the examples above. However, the advantage of sc/snRNA-seq for genes and TFs discovery and dynamics of regulation cannot be assessed but only by in vitro or in vivo approaches.

6. Perspectives

The study of the SAM is a topic that has captivated humanity historically. Initially, based on anatomical approaches, the prevailing evolutive notion was that the simple meristems composed of one or a few cells were the most primitive, with increased complexity during plant evolution. However, our current understanding suggests that the SAM has multiple origins and that meristems with an apical cell may reappear in vascular clades from ancestors with multicellular meristems [2]. However, further evidence is needed to confirm and specify the multiple origins of SAM. Among all groups of plants, angiosperms are the most studied. However, research into the conservation of SAM regulatory pathways is still in its early stages.

Angiosperm meristems, mainly dicot meristems, have been extensively studied, and there is an increasing interest in deciphering if SAM regulatory pathways are conserved among diverse groups of plants. Evidence suggests that monocot and dicot meristems could exhibit distinct regulatory loops. However, if such diversity exists within angiosperms, one might wonder about its prevalence in other plant groups. For instance, does research on meristem regulation in bryophytes and lycophytes need to commence without assuming similar regulatory mechanisms?

Significant progress has been made in understanding the maize SAM through single-cell and spatial transcriptomics. However, developing this technique in non-model species remains challenging mainly for three reasons. First, protocols for sc/snRNA-seq in plants are species-dependent. Second, developing these techniques on meristems with only one apical cell is challenging as the cell recovery on the overall efficiency of current scRNA-seq protocols can vary between <1% to >60% across cells, depending on the method used [140]. Therefore, ensuring the representation of a sufficient percentage of apical cells would necessitate adept technical skills. Third, as sc/snRNA-seq analyses rely on gene markers to identify clusters, the poor genomic information available for non-model species will limit their significance. This will also be impacted by the lack of information regarding cellular domains of the SAM and gene expression and regulatory pathways involved in regulation. Therefore, all this information must be generated in advance.

Comparative studies between multiple species have the potential to shed light on the regulatory pathways of the SAM, help clarify its origin, and address related questions. For instance, are there other regulatory cycles besides the CLV-WUS? Do they act in a concerted way or independently? Or are they species-specific? That would help explore the presence of conserved or non-conserved regulatory loops in monocots and other plant groups. Furthermore, it will be essential to investigate whether the expansion of SAM regulatory genes relates to plant morphological traits across plant phylogeny. Another exciting perspective is to search for conserved mechanisms that promote and maintain cell dedifferentiation and proliferation, which would have a great impact on different fields of research.