Male Germ Cell Specification in Plants

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


Introduction
Generally, multicellular organisms initiate their life cycle from a single totipotent cell.As cell division progresses and ordered multicellular organization ensues, certain cells are specified as germ cells (Table 1, glossary), responsible for transmitting genetic information from one generation to the next.In most higher animals, the germline (Table 1) is segregated early during embryogenesis [1].Primordial germ cells (PGCs), distinguished from somatic cells (Table 1), serve as bipotential precursors of eggs and sperms, developing into eggs in the ovary and sperm in the testes [1,2].Unlike animals, the architecture of higher plants primarily arises from post-embryonic development.In plants, it is generally understood that the germline is segregated late during development post-embryonically and tightly coupled with organogenesis.In comparison to animals, the embryogenesis of plants is relatively rudimentary.During embryogenesis, the basic apical-basal axis is established, characterized by the formation of the shoot apical meristem (SAM) and root apical meristem (RAM) at opposite poles of the embryo.Subsequently, during postembryonic development, the SAM and RAM give rise to the entire aerial and underground structures, respectively.
In a representative dicot, Arabidopsis, SAM precursor cells are initially distinguishable in dermatogen-stage (16-cell) embryos [3] (Figure 1).During the vegetative development phase, the SAM generates leaves at its periphery.At the boundary between the SAM and leaf primordia, a group of cells are 'detached' from the SAM and sequestered earlier from the surroundings, undergoing minimal cell division [4] (Figure 1).These initial axillary meristem (AM) precursors express SHOOT MERISTEMLESS (STM) and, through the activation of WUSCHEL(WUS) and CLAVATA 3 (CLV3), give rise to new functional SAMs, thereby generating new branches [5,6].This process recurs throughout the plant body, contributing to the formation of a complex architecture [7].During the reproductive phase, the developmental identity of the SAM transitions to the inflorescence meristem (IM), accompanied by changes in its doming shape [8] and gene regulatory network (GRN) [9] (Figure 1).In Arabidopsis, similar to the process of AM formation, the floral meristem (FM) initiates at the axis of cryptic bract primordia (Figure 1).The key factors in meristem maintenance, namely, WUS and CLV3, are reactivated at the center of the FM to sustain meristematic activity, leading to the initiation of floral organs in four concentric whorls (from outer to inner: sepals, petals, stamens, and carpels) (Figure 1).Male and female germ cells are progressively specified within specific inner tissues of the stamens and carpels, indicating the close link between germ cell specification and the morphogenesis of reproductive organs.In this paper, we will review our current understanding of male germ cell specification in plants, primarily using Arabidopsis as an example.

Germ cell:
A germ cell, also known as a gamete precursor cell or reproductive cell, typically arises from PGCs during early embryonic development in animals.Germ cells undergo meiosis to form gametes (sperm or eggs) in animals and plants, delivering genetic information from one generation to their offspring.Germline: A line of cells that separate from somatic cells and become germ cells.Primordial germ cell: A primordial germ cell (PGC) is a specialized cell type that gives rise to gametes (sperm and eggs) in animals during embryonic development.These cells are set aside early in embryonic development and undergo a unique series of developmental events that ultimately lead to their differentiation into mature gametes.Somatic cells: Cells in the organism that are not germ cells.Archesporial cell (AR cell): These cells give rise to the spore mother cell, which will further undergo meiosis to produce spores in plants.AR cells are specified early within the floral meristem or sporophytic tissue, which could be analogous to animal PGCs.Germ cell lineage: The cells that give rise to the gametes, including the primordial germ cells, the developing eggs and sperms, and the mature gametes.Morphogen: A morphogen is a signaling molecule that diffuses through tissues and forms a concentration gradient, providing positional information to cells and directing their differentiation and patterning during development.

Pattern Formation: Spatial-Temporal Initiation of Stamen Primordia
Different from animals, plant cells cannot move, and thus, germ cell specification must be tightly coordinated with organogenesis.In flowering plants, male germ cells are embedded in the male reproductive organ-the stamen.The number of stamens varies among species, from a few to dozens.In Arabidopsis, there are six stamens containing a distal structure, called the anther (where germ cells are located), and a proximal structure, called the filament (responsible for supporting and transporting nutrients).In the wildtype floral bud, four medial (long) stamen primordia arise initially along the periphery of the FM, with two lateral (short) stamen primordia initiating later [10] (Figure 2, a).The underlying mechanism governing the synchronization of the timing and positional development of stamen primordia remains unclear.
All lateral organs originate from the shoot apical meristem or its derivates (i.e., axillary meristem and floral meristem).Physical (e.g., size, shape, mechanical properties) and biochemical factors (e.g., phytohormones) are widely incorporated in the regulation

Stamen Morphogenesis 2.1. Pattern Formation: Spatial-Temporal Initiation of Stamen Primordia
Different from animals, plant cells cannot move, and thus, germ cell specification must be tightly coordinated with organogenesis.In flowering plants, male germ cells are embedded in the male reproductive organ-the stamen.The number of stamens varies among species, from a few to dozens.In Arabidopsis, there are six stamens containing a distal structure, called the anther (where germ cells are located), and a proximal structure, called the filament (responsible for supporting and transporting nutrients).In the wild-type floral bud, four medial (long) stamen primordia arise initially along the periphery of the FM, with two lateral (short) stamen primordia initiating later [10] (Figure 2a).The underlying mechanism governing the synchronization of the timing and positional development of stamen primordia remains unclear.
All lateral organs originate from the shoot apical meristem or its derivates (i.e., axillary meristem and floral meristem).Physical (e.g., size, shape, mechanical properties) and biochemical factors (e.g., phytohormones) are widely incorporated in the regulation of the number and spatial-temporal distribution of leaves and floral buds at the shoot apical meristem [11][12][13][14][15][16].Several pieces of evidence suggest that a similar mechanism may also apply to floral organogenesis.Indeed, dynamic auxin distribution and auxin biosynthesis are both crucial for regulating floral organ number and identity [17,18].In addition, a recent study by Zhu and colleagues revealed that the timing of sepal initiation is regulated by DEVELOPMENT RELATED MYB-LIKE 1 (DRMY1) via focusing the auxin and cytokinin signaling pattern [19].This mechanism is very similar to the robustness of the phyllotactic pattern control at the SAM, underscoring the importance of two distinct hormone-based fields for achieving robust pattern formation [14,20].
Meristem size and activity play significant roles in organ number control.For instance, WIGGUM (WIG) functions in parallel with other genes like CLAVATA (CLV) to regulate meristem structure and floral organ number [21].Conversely, mutations in PERIANTHIA (PAN) result in increased sepal and petal numbers but decreased stamen numbers, independent of the floral meristem size and floral identity genes [22].Another boundary gene, SUPERMAN (SUP), expressed at the boundary between whorl 3 and whorl 4, is responsible for floral meristem termination and the repression of stamen primordia's initiation from whorl 4 [23] through fine-tuning local auxin biosynthesis via interaction with polycomb repressive complex 2 (PRC2) [24].Although auxin is incorporated in the process of stamen initiation, whether stamen primordia follow a specific phyllotactic pattern and whether physical factors participate in regulating stamen initiation remain largely unexplored.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 4 of 14 whorl 4, is responsible for floral meristem termination and the repression of stamen primordia's initiation from whorl 4 [23] through fine-tuning local auxin biosynthesis via interaction with polycomb repressive complex 2 (PRC2) [24].Although auxin is incorporated in the process of stamen initiation, whether stamen primordia follow a specific phyllotactic pattern and whether physical factors participate in regulating stamen initiation remain largely unexplored.L1, the outermost cell layer; L2-d, cells derived from the second layer; PPC, primary parietal cell; Ar, archesporial cell; SPC, secondary parietal cell; PSC, primary sporogenous cell; E, epidermis; En, endothecium; ML, middle layer; T, tapetum.Images are taken from PNAS [25] with permission.

Stamen Polarity: Establishment of three AXES
Once bulging out from the meristem, the stamen primordia quickly adopt a threedimensional structure with three axes: adaxial-abaxial, medial-lateral, and proximaldistal (Figure 2a).Similar to other lateral organs, stamens are believed to originate from leaves [26].
Adaxial-abaxial axis.In Arabidopsis, leaf primordia initiate at the flank of the SAM with a slightly ellipsoid shape.However, leaf polarity is determined much earlier than the L1, the outermost cell layer; L2-d, cells derived from the second layer; PPC, primary parietal cell; Ar, archesporial cell; SPC, secondary parietal cell; PSC, primary sporogenous cell; E, epidermis; En, endothecium; ML, middle layer; T, tapetum.Images are taken from PNAS [25] with permission.

Stamen Polarity: Establishment of Three AXES
Once bulging out from the meristem, the stamen primordia quickly adopt a threedimensional structure with three axes: adaxial-abaxial, medial-lateral, and proximaldistal (Figure 2a).Similar to other lateral organs, stamens are believed to originate from leaves [26].
Adaxial-abaxial axis.In Arabidopsis, leaf primordia initiate at the flank of the SAM with a slightly ellipsoid shape.However, leaf polarity is determined much earlier than the initial outgrowth by a group of factors such as auxin, small RNAs, HD-ZIP III family proteins, ASYMMETRIC LEAVES 2 (AS2), and KANADI (KAN) genes.These factors interact and sequentially define the adaxial-abaxial polarities (also called dorsoventrality) and the boundaries in between [27][28][29][30][31][32][33].Once leaf primordia bulge out, a microtubule-mediated mechanical feedback mechanism is proposed to sustain or even amplify the leaf flatness [34].
In contrast to leaves, stamen primordia initiate in the inner third whorl of the floral meristem (FM) with approximately 28 cells in the initium, based on live imaging observation [35].The shape of anther primordia is initially isotropic, resembling a radially symmetric disc from a cross-section view [36,37].Around four days after initiation, the distal part of stamen primordia begins to grow more isotropically, corresponding to the formation of the anther (where germ cells will reside in) [35].Subsequently, from a transverse section view, the shape of the anther shifts from a disk to a trapezoid (from around anther stage 2) and finally to a butterfly shape by anther stage 5 [36,37].This symmetry transition is documented in rice anthers, where the polarity genes Oryza sativa ETTIN1 (OsETT1) (Auxin Response Factor, ARF gene) and PHB3 (DH-ZIP III family gene) undergo a shift in gene expression.OsETT1 is initially expressed on the abaxial side of anthers, and later on, it is located at both the adaxial and abaxial sides.By contrast, PHB3 expression shifts from the adaxial side to the intermediate domain of the adaxial and abaxial anther lobes [38].Similarly, symmetry transition in Canna indica anthers is reported to be correlated with the rearrangement of CiPHB (adaxial gene) and CiFIL (abaxial gene) [39].In Arabidopsis flowers, PHB expression initially occurs on the adaxial side but later becomes restricted to the two lateral domains of anther primordia [40], which is very similar to what has been found in rice anthers [38].By contrast, ETTIN (ETT) is initially expressed in the abaxial domain of anther primordia; however, at least around stages 4 and 5, ETT is abundant at the procambial strand and inner borders of locules [41].This expression pattern is very different from the FIL expression pattern, which, along with other YABBY genes (YABBY2 and YABBY3), is localized abaxially in anther primordia until at least the butterfly shape of the anther is formed [42].While the genetic manipulation of adaxial-abaxial gene expression in leaves can generate four-bladed leaves, mimicking rice anthers [29], the dynamics of dorsoventrality in Arabidopsis anthers require further elucidation.
Medial-lateral axis.Another aspect of three-dimensional organ shape is along the medial-lateral direction.During leaf development, medial-lateral polarity is mediated by two WUSCHEL-RELATED HOMEOBOX (WOX) family genes, WOX1 and PRESSED FLOWER (PRS).These genes are conserved in several flowering plants and play a role in controlling leaf and many lateral organ expansions [28,29,[43][44][45][46].While PRS is involved in sepal development [28,47], the petals and stamens are fairly normal in prs [47].
Proximal-distal axis.Proximal-distal polarity is established as the stamen primordia grow away from the floral meristem, resulting in a distal anther with bilateral symmetry and a proximal filament with radial symmetry.However, in some basal taxa, the filaments maintain the adaxial-abaxial polarity, exhibiting a flat structure [48].The distinct polarity control along the proximal-distal axis is still elusive.In leaf development, BLADE-ONPETIOLE 1 and 2 (BOP1/2) are well-known factors controlling the specification of the leaf petiole domain.In bop1 bop2 mutants, the petiole domain is diminished and is overtaken by an extra blade, whereas the anther and filament development remain largely unaffected [49].In Arabidopsis stamens, NUBBIN (NUB) and JAGGED (JAG) redundantly regulate stamen proximal-distal polarity.In nub jag mutants, distal anther development is severely disrupted, sometimes resulting in an abaxialized radial symmetric distal struc-ture [40].In fact, while the shifting of dorsoventrality is necessary for the establishment of anther shape in rice, it is assumed that the adaxial polarity might be lost in the proximal part of the stamen, leading to an abaxialized filament [38].The fact that anthers exhibit a radialized shape in certain polarity mutants, such as kan, phb-1d, and fil [38,[50][51][52][53], indicates that the adaxial-abaxial polarity might also play a role in coordinating proximal-distal axial specification.

Growth Dynamics of Anthers
The most prominent function of the anther is to produce male germ cells.Based on the anther shape and size, the staging for early anther development has been elaborately described [36,37,54,55].Recently, taking advantage of live imaging and quantitative image analyses, Silveira and colleagues provided detailed insights into stamen morphodynamics in Arabidopsis [35].Along the proximal-distal axis, rapid growth initially occurs at the tip of anther primordia, corresponding to the differentiation of the anther and filament.Subsequently, growth ceases, also starting from the anther tip.This basipetal differentiation pattern is reminiscent of what Gould and Lord proposed in lily anther: there is a growth wave from the tip to the base that could be correlated with auxin flow [56], although such a growth wave is not observed in maize anthers [57].Another difference in the anther growth pattern between Arabidopsis and maize is along the adaxial-abaxial axis.While the development of anther lobes is highly canalized and synchronized in maize [58], it is proposed to be temporally different in Arabidopsis, possibly due to variations in the growth rates between the adaxial and abaxial lobes [35].Along the medial-lateral axis, the growth rate is higher in the lateral domain.This growth pattern is fascinating, suggesting that heterogeneous growth might be relevant to the internal tissue growth of the anther lobe [35].

Concentric Layered Structure and GC Formation
It has been widely accepted that male germ cells are initiated from L2 cells in the stamen primordia, whereas their traceability back to the shoot apical meristem remains hypothetical [55,[58][59][60][61].The initial form of a germ cell in the anther, known as the archesporial cell (AR, Table 1), is characterized by its large size and nucleus [25,37,55].After several rounds of mitosis, these ARs become mature to form so-called pollen mother cells (PMCs) or microsporocytes, which ultimately undergo meiosis.As the germ cell lineage (Table 1) is specified, surrounding somatic cells divide in an orderly manner and form a concentric layered structure.In each somatic layer, the cells adopt a unique cell fate, including the tapetum, middle layer, endothecium, and epidermis from the innermost to the outermost layer (Figure 2b).The number of each type of somatic cell layer may vary among different species [48].
Two different models are proposed to explain the formation of these concentric multilayer structures.In the lineage model, ARs localized at L2 are proposed to divide asymmetrically, with the smaller daughter cell generating the outer somatic layers and interior larger daughter cells serving as germinal lineage founders.In contrast, the positional model suggests that there is no clear enlargement of hypodermal ARs and no subsequent asymmetric division in maize anthers.Instead, the ARs are proposed to initiate from the innermost of a small group of L2-derived cells with a population of around 100 [57].While these differences may probably be species-dependent, among all the somatic cell types, the tapetum appears to be present and conserved at least across flowering plants, with the function of nursing the germ cells [48,61].By contrast, the function of the middle layer remains unclear [61][62][63], and its presence varies among species [48].Advances in imaging techniques, single-cell sequencing, and spatial transcriptomics are expected to help us find more factors responsible for the specification of different cell types, as well as their interactions and functionalities (e.g., [62,64,65]).

Germ Cell Specification: Molecular Players
In the past few decades, plenty of factors involved in regulating germ cell fate determination have been identified (for comprehensive reviews, see [37,55,61,63,[66][67][68]).In the gene regulatory network (GRN), SPROCYTLESS/NOZZLE (SPL/NZZ) emerges as a central hub.SPL/NZZ was first identified in Arabidopsis, playing a pivotal role in specifying both male and female germ cells [69,70], and its homologs have been recently identified in tomato, rice, and cucumber [71][72][73][74].Interestingly, while it is suggested that there is no meristem in anthers [55], the SPL/NZZ-centered circuit in germ cell specification bears striking similarities to the WUS-centered circuit in regulating shoot apical meristem activity (Figure 3).In these circuits, a group of factors like leucine-rich repeat receptor-like protein kinases (LRR-RLKs), mitogen-activated protein kinases, hypoxia, and auxin are involved in forming complex feedback loops (Figure 3).In this section, we will briefly compare the similarities between the regulation of SAM homeostasis and the specification of GC-specific processes.

Discussion and Perspectives
Understanding how germ cells are originally specified is a captivating question in developmental biology.It has been a long-lasting notion that plants and animals employ different strategies to establish their germlines.However, recent research suggests that this may not always be the case.For instance, in the majority of animal classes, the germline segregates after embryogenesis, a process very similar to what occurs in plants [104].By contrast, in vertebrates and ecdysozoans, germline segregation, as commonly observed, occurs during embryogenesis [104].One explanation for these differences might be motility: if an organism is more motile, like vertebrates and ecdysozoans, it is much easier to shield the germline from fluctuating environments.Conversely, if the organism is motile-limited or even sessile, like some lower animals and plants, germline segregation may be reasonably delayed or more tunable to buffer against environmental fluctuations.For example, cnidarians (corals and jellyfish) possess high regenerative ability and do not succumb to age.These regenerative abilities and plant-like clonal growth rely on a group of adult stem cells called i-cells, which are proven to have the capability to differentiate into all somatic lineages and germ cells [105].
In flowering plants, much like animal stem cells, groups of meristematic cells are found in the shoot apex, root apex, and also the stem [106].Within the shoot apical meristem, a CLV-WUS feedback loop maintains meristem homeostasis, balancing cell selfrenewal and differentiation to orchestrate all areal organ formation (including determinate lateral organs and indeterminate new branches).Interestingly, while anthers are not typically considered to possess a meristem [55], a SPL/NZZ-based circuit that determines the germ cell specification in Arabidopsis anther lobes closely resembles the CLV-WUS-based circuit structure regulating SAM activity (Figure 3).This suggests there might be a very tight link between meristematic cells and germ cells in plants.This notion is reinforced by recent research indicating that two ARGONAUTE (AGO) proteins (AGO5 and AGO9) mark both meristematic cells in the SAM and GCs in reproductive organs, LRR-RLKs are key factors participating in a wide range of developmental processes.Among them, BARELY ANY MERISTEM 1/2/3 (BAM1/2/3), CLAVATA3 INSENSITIVE RECEPTOR KINASES (CIKs), and RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2) are well known in regulating germ cell specification and meristem activity [75][76][77][78].Plant RLKs share structural similarities with animal receptor tyrosine kinase (RTK) and exhibit similar downstream components, such as the MAPK cascade and ROS production [79].In the shoot apical meristem, MPK3/6 are activated by CLV3 signals through RLKs (mainly CLV1 and BAM1) to regulate meristem homeostasis by repressing WUS expression [80], while MPK3/6 can stabilize SPL/NZZ by direct phosphorylation to ensure germ cell specification [81].
In animals, the maintenance of the pluripotent state of stem cells requires hypoxic conditions, whereas higher oxygen tension promotes cell differentiation [82].In the plant SAM, there is an increase in the oxygen gradient from the inner to outer layers.The inhibition of the hypoxic response by exposing plantlets to hyperoxic conditions impairs meristem activity [83].Interestingly, the redox status has also been implicated as a determinant for germ cell specification in maize anthers [84].The hypoxia status in maize anthers is regulated by the glutaredoxin gene MALE STERILE CONVERTED ANTHER 1 (MSCA1) [84].While the redox status in other plant anthers has not been experimentally measured, the mutation of MSCA1 homolog genes in Arabidopsis and rice also leads to defects in germ cell formation [85,86], suggesting a plausible conservative function of hypoxia in germ cell specification at least among angiosperms.
It has long been noticed that auxin and its related signaling components are present at the summit and inner core of the SAM [87,88].Recent evidence indicates that auxin biosynthesis and transport are essential for WUS expression, and WUS, in turn, rheostatically controls the auxin signaling pathway via epigenetic regulations [89,90].Similar to this WUS-auxin feedback, SPL/NZZ-auxin feedback has recently been identified in Arabidopsis anthers, where TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1)/TRYPTOPHAN AMINOTRANSFERASE RELATED 2 (TAR2)-mediated auxin biosynthesis is crucial for the centripetal auxin distribution pattern and GC specification by the activation of SPL/NZZ expression.SPL/NZZ, in turn, mediates the effect of auxin on GC specification and modifies auxin biosynthesis to maintain a centripetal auxin distribution [25].Apart from auxin, brassinosteroid (BR) signaling has also been implicated in regulating SPL/NZZ expression and thus GC formation [91][92][93].However, while BRs are important for organ boundary formation in the SAM, their functions in the central meristem remain unclear [94].
Despite the intriguing parallels between SAM homeostasis and germ cell-specific regulation, there exists a physical separation of the SAM and anther lobes in space and time.In Arabidopsis, the homeotic gene AGAMOUS (AG) plays a crucial role in bridging the gap (Figure 3).As a bifunctional transcription factor, WUS acts as a repressor in regulating the cell stemness in the SAM and as an activator in regulating floral patterning via inducing AG expression [95].Once activated in the floral meristem (FM), AG in turn represses WUS by activating KNUCKLES (KNU) transcription [96][97][98][99].In ag mutants, all floral organs are transformed into sepals and petals, with no germ cell formation.The ectopic expression of AG in ag directly activates SPL/NZZ transcription, hence inducing germ cell formation in petals [100].Moreover, with the accumulation of AG, the petals in ag flowers can ultimately be transformed into stamens [100,101].This indicates that AG is also a factor bridging anther morphogenesis and germ cell specification.
In fact, accumulating evidence is beginning to reveal how anther formation may be linked to its function as a germ cell container at the molecular level.For example, recent work by Zheng and colleagues demonstrated that auxin, a morphogen (Table 1) in plants, acts as a key factor in guiding male germ cell specification in Arabidopsis anthers [25].This echoes the involvement of many factors that regulate organ polarity, which is also auxin-related, that also mediate germ cell formation by interacting with SPL/NZZ (e.g., YAABY, ETTIN) [102,103].

Discussion and Perspectives
Understanding how germ cells are originally specified is a captivating question in developmental biology.It has been a long-lasting notion that plants and animals employ different strategies to establish their germlines.However, recent research suggests that this may not always be the case.For instance, in the majority of animal classes, the germline segregates after embryogenesis, a process very similar to what occurs in plants [104].By contrast, in vertebrates and ecdysozoans, germline segregation, as commonly observed, occurs during embryogenesis [104].One explanation for these differences might be motility: if an organism is more motile, like vertebrates and ecdysozoans, it is much easier to shield the germline from fluctuating environments.Conversely, if the organism is motilelimited or even sessile, like some lower animals and plants, germline segregation may be reasonably delayed or more tunable to buffer against environmental fluctuations.For example, cnidarians (corals and jellyfish) possess high regenerative ability and do not succumb to age.These regenerative abilities and plant-like clonal growth rely on a group of adult stem cells called i-cells, which are proven to have the capability to differentiate into all somatic lineages and germ cells [105].
In flowering plants, much like animal stem cells, groups of meristematic cells are found in the shoot apex, root apex, and also the stem [106].Within the shoot apical meristem, a CLV-WUS feedback loop maintains meristem homeostasis, balancing cell self-renewal and differentiation to orchestrate all areal organ formation (including determinate lateral organs and indeterminate new branches).Interestingly, while anthers are not typically considered to possess a meristem [55], a SPL/NZZ-based circuit that determines the germ cell specification in Arabidopsis anther lobes closely resembles the CLV-WUS-based circuit structure regulating SAM activity (Figure 3).This suggests there might be a very tight link between meristematic cells and germ cells in plants.This notion is reinforced by recent research indicating that two ARGONAUTE (AGO) proteins (AGO5 and AGO9) mark both meristematic cells in the SAM and GCs in reproductive organs, implying a protective function of AGOs in safeguarding the genome from damage by transposons [107].Thus, while plants and animals diverged early in evolutionary history [108], they may have independently evolved similar mechanisms to preserve germ cell potential in meristematic or stem cells, where the mutation rate in the genome is relatively low [4,107,109].
While this comparison is intriguing, several questions remain.For instance, evolutionarily, the function of WUS appears later than that of CLVs.Thus, CLV-cytokinin cross-talk and its mediated auxin homeostasis have recently been proposed to be a more ancient property for SAM homeostasis [110][111][112].Additionally, the homologs of SPL/NZZ have only been identified in angiosperms.It remains unknown whether SPL/NZZ-like proteins exist in other plant species, particularly early land plants like bryophytes.Moreover, auxin has been proven to be essential for the development of meristem function in Marchantia [113,114].However, the function of auxin in germ cell specification in bryophytes is not clear.What could be the simplest circuit conserved evolutionarily for germ cell induction and probably also for meristematic cell fate acquisition is an open question.
Another interesting factor that commonly guides germ cell specification in both animals and plants is morphogen (Table 1).In animals, retinoic acid (RA) exhibits properties characteristic of morphogen by providing positional information to cells and directing their differentiation and patterning during animal development.RA concentration gradients play a critical role in coordinating complex developmental processes and ensuring the proper formation of tissues and organs in various animal species, which is very comparable to the function of auxin in plant development [115,116].Notably, during the development of the reproductive system, retinoic acid is essential for the formation of gonads and germ cells in various animal models [117][118][119].This parallels the role of auxin in germ cell specification in Arabidopsis anthers [25].
The last but not least interesting question is the coordination of germ cell specification in plants.In Brassicaceae, for instance, the proportion of flowers (harboring germ cells) initiated on secondary inflorescences (transformed from the axillary meristem) represents around half of the total floral production, and secondary inflorescences continue to mutually inhibit each other.Walker and Bennett hypothesized that this phenomenon can be explained by the extension of the canalization model for dominance and/or shoot branching [120], which is known to be regulated by auxin transport.Additionally, this coordination might be associated with the depletion of meristematic potential at the shoot apical meristem.Since developmental timing in plants is proposed to be strongly dependent on growth [121], auxin, as a key factor of both long-distance and local signals in orchestrating plant growth and morphogenesis, might be a good candidate for further investigation into its potential role in coordinating germ cell specification throughout the plant.With the rapid advancement of cutting-edge imaging techniques, computational modeling, and the emergence of novel model systems (e.g., [122]), we are poised to gain a deeper understanding of how plants specify their germ cells.

Figure 2 .
Figure 2. Anther development in the model plant Arabidopsis.(a) The successive developmental stages of stamens in Arabidopsis flowers.White arrow heads indicate the anthers at the distal part of stamen primordia.Scale bars, 50 µm.(b) A schematic representation of the germ cell specification process in Arabidopsis anthers.(Left) A cross-section of the anther.(Right) The evolution of germ cell specification in an anther lobe at different stages.Cell types are marked out with different colors.L1, the outermost cell layer; L2-d, cells derived from the second layer; PPC, primary parietal cell; Ar, archesporial cell; SPC, secondary parietal cell; PSC, primary sporogenous cell; E, epidermis; En, endothecium; ML, middle layer; T, tapetum.Images are taken from PNAS[25] with permission.

Figure 2 .
Figure 2. Anther development in the model plant Arabidopsis.(a) The successive developmental stages of stamens in Arabidopsis flowers.White arrow heads indicate the anthers at the distal part of stamen primordia.Scale bars, 50 µm.(b) A schematic representation of the germ cell specification process in Arabidopsis anthers.(Left) A cross-section of the anther.(Right) The evolution of germ cell specification in an anther lobe at different stages.Cell types are marked out with different colors.L1, the outermost cell layer; L2-d, cells derived from the second layer; PPC, primary parietal cell; Ar, archesporial cell; SPC, secondary parietal cell; PSC, primary sporogenous cell; E, epidermis; En, endothecium; ML, middle layer; T, tapetum.Images are taken from PNAS[25] with permission.

14 Figure 3 .
Figure 3. Gene regulatory circuits that regulate meristem cell homeostasis and germ cell specification.Red circles indicate the hypoxia gradients.

Figure 3 .
Figure 3. Gene regulatory circuits that regulate meristem cell homeostasis and germ cell specification.Red circles indicate the hypoxia gradients.