2. SAM structure and organogenic activity

Phyllotaxis is an outcome of meristem organogenic activity; thus, SAM morphology is important for understanding pattern formation. The size and shape of the gymnosperm meristem varies from small and narrow conical apices, for example in Cupressus, Thuja, Juniperus or Araucaria, reaching ca. 120 μm (or less) in diameter and ca. 50–70 μm in height (Crafts, Reference Crafts1943b; Griffith, Reference Griffith1952; Pillai, Reference Pillai1963; Pillai, Reference Pillai1963a, Reference Pillai1964) to relatively huge, broad dome-like or even flat, 300–400 μm in diameter, as in Pinus, Pseudotsuga, Torreya (Crafts, Reference Crafts1943b; Kemp, Reference Kemp1943). The biggest meristems, exceeding even 3,000 μm in diameter, occur in cycads (Foster, Reference Foster1940, Reference Foster1943; Johnson, Reference Johnson1945). Importantly, depending on the meristem’s divisional and organogenic activity, its size and shape show seasonal variations in some species (Curtis & Popham, Reference Curtis and Popham1972; Kemp, Reference Kemp1943; Owens, Reference Owens1984; Owens et al., Reference Owens, Molder and Langer1977; Owens & Molder, Reference Owens and Molder1973b, Reference Owens and Molder1976; Parke, Reference Parke1959; Pillai, Reference Pillai1963a, Reference Pillai1963b, Reference Pillai1964; Pillai & Chacko, Reference Pillai and Chacko1978; Romberger, Reference Romberger1963; Sacher, Reference Sacher1954; Sterling, Reference Sterling1946b).

The SAM structure in gymnosperms seems to be relatively less complicated than in angiosperms due to the presence of only one superficial group of initial cells (Evert, Reference Evert2007; Romberger, Reference Romberger1963). Such a group usually contains a few impermanent apical initial cells, which could be replaced during ontogeny (Conway & Drinnan, Reference Conway and Drinnan2017; Ruth et al., Reference Ruth, Klekowski and Stein1985; Zagórska-Marek & Turzańska, Reference Zagórska-Marek and Turzańska2000). They occupy the central part of the meristem, forming the apical initial layer and divide in both planes: perpendicular (anticlinal division) and parallel (periclinal division) to the meristem surface (Figure 1). Anticlinal divisions of the initial cells provide cells to the peripheral zone (PZ), whose outer region is involved in organ primordia initiation, while the inner region is responsible for vascular meristem (procambium) development. On the other hand, periclinal divisions of the superficial initial cells supply cells to the central mother cell (CMC) zone, corresponding to the organising centre of the angiosperm SAM (Bäurle & Laux, Reference Bäurle and Laux2005; Kean-Galeno et al., Reference Kean-Galeno, Lopez-Arredondo and Herrera-Estrella2024; Kwiatkowska, Reference Kwiatkowska2008). This zone is characterised by a slower rate of cell divisions and thus a prolonged cell cycle. The CMC zone is delimited from the rib meristem, located below, by a narrow transition zone of intensively dividing cells, which extends to the PZ and can reach the meristem surface (Figure 1). Generally, the zonation pattern of the gymnosperm apex is distinguishable due to such pronounced cytohistological differences that they were described for the first time in this plant group (not in angiosperms!) (Gifford & Wetmore, Reference Gifford and Wetmore1957). However, the distinctiveness of particular zones varies depending on the genus, apex shape and size, frequency of periclinal divisions and ontogenetic stage, and can be seasonally changeable. For instance, the well-defined zonation, mentioned above, was described in Ginkgo (Foster, Reference Foster1938; Gunckel & Wetmore, Reference Gunckel and Wetmore1946a; Romberger, Reference Romberger1963), and similar, though less pronounced patterning is visible, for example, in cycads and the Pinaceae family (Cross, Reference Cross1943; Foster, Reference Foster1939, Reference Foster1940, Reference Foster1943; Johnson, Reference Johnson1939, Reference Johnson1945; Kemp, Reference Kemp1943; Sacher, Reference Sacher1954; Sterling, Reference Sterling1945a, Reference Sterling1945b). Some genera from the Taxaceae and Cupressaceae have a discrete layer of superficial cells (so-called ‘pseudotunica’) formed due to limited periclinal divisions within the PZ. Extremely rare periclinal divisions in superficial cells of the adult SAMs of the Araucariaceae (and possibly in some Podocarpaceae and Gnetaceae) cause the structure of their meristems to resemble a tunica-corpus organisation of angiosperms (Cross, Reference Cross1942; Griffith, Reference Griffith1952; Johnson, Reference Johnson1950; Pillai, Reference Pillai1964; Romberger, Reference Romberger1963; Romberger et al., Reference Romberger, Hejnowicz and Hill1993; Sacher, Reference Sacher1954; Sterling, Reference Sterling1958).

Figure 1.

Schemes of the shoot apical meristem (SAM) structure in gymnosperms (a) and angiosperms (b). Initial cells for a superficial group of apical initials in gymnosperms and for three groups of initials in angiosperms are drawn in green. In gymnosperms, initial cells divide anticlinally and periclinally, while in angiosperms divisions in two outer groups of initials (in tunica) are anticlinal and in the inner group of initials (in corpus) anticlinal and periclinal. Central mother cell zone (CMC) and organising centre (OC) are labelled in yellow, peripheral zone in blue. Light grey colour marks the rib meristem; dark grey (in gymnosperm) shows the transition zone.

Figure 1 Long description

Initial cells for a superficial group of apical initials in gymnosperms and for three groups of initials in angiosperms are drawn in green. In gymnosperms, initial cells divide anticlinally and periclinally, while in angiosperms divisions in two outer groups of initials (in tunica) are anticlinal and in the inner group of initials (in corpus) anticlinal and periclinal. Central mother cell zone (CMC) and organizing center (OC) are labeled in yellow, peripheral zone in blue. Light grey color marks the rib meristem; dark grey (in gymnosperm) shows the transition zone.

Lateral organ initiation starts in the PZ, by divisions of subsuperficial cells. The shoot vegetative meristem produces primordia of different identity, assimilating leaves and scales (cataphylls), while the reproductive apex initiates bracts (bract scales), seed (ovuliferous) or microsporangia-bearing scales and forms a strobilus or cone (Romberger et al., Reference Romberger, Hejnowicz and Hill1993).

As gymnosperms are long-lived trees or shrubs, composed of numerous branches subordinated to the main axis (monopodial branching), the SAM on each axis (branch) functions for many years, showing seasonal changes in its activity, size and shape. During the dormant period, the meristem ceases its activity and is usually covered by scales forming a bud. The meristem hidden within the bud can bear the leaf primordia, initiated during the prior vegetative season, which, however, will develop in the subsequent year (so-called predetermined growth). The axils of some leaf and/or scale primordia can contain initiated and partially developed axillary buds (Wenzel et al., Reference Wenzel, Xia, Saunders, Ly, Mo and Holloway2024, Reference Wenzel, Mo, Chan, Xia, Westgate, Saunders, Ly and Holloway2026). As a whole, such a structure is treated as an embryonic (preformed) shoot with extremely shortened internodes (Romberger, Reference Romberger1963; Sterling, Reference Sterling1945a, Reference Sterling1958). After bud bursting, scales detach and the preformed shoot rapidly elongates. Then, the meristem resumes its divisional and organogenic activity and produces first the scales, which will form the bud, and finally leaves for the next vegetative season. Thus, along one continuously growing branch, alternating zones with assimilating leaves and short fragments covered with scars of scale leaves (seasonal borders) are present, being the outcomes of seasonal activity of meristem (Parke, Reference Parke1959; Romberger, Reference Romberger1963; Romberger et al., Reference Romberger, Hejnowicz and Hill1993; Zagórska-Marek, Reference Zagórska-Marek1985). In some species, for example in Abies concolor, Pinus lamberghiana, P. ponderosa, Pseudotsuga taxifolia, Picea abies, this sequence of lateral organ initiation is modified, and scale initiation takes place before or during the embryonic shoot elongation (Parke, Reference Parke1959; Romberger, Reference Romberger1963; Sacher, Reference Sacher1954; Sterling, Reference Sterling1946b; Wenzel et al., Reference Wenzel, Xia, Saunders, Ly, Mo and Holloway2024). Alternatively, in the Cupressaceae, the preformed, embryonic shoot and dormant bud are not formed. Meristem activity and organogenesis are temporally arrested under unfavourable environmental conditions and resumed afterwards (Stankova et al., Reference Stankova, Stankov and Panetsos1999).

In the majority of gymnosperms (with the exception of Pinus), below the bud, in the central part of the stem, the collenchyma plate (crown) is formed. It separates the meristem from the mature tissues (mostly those of the pith) and is composed of a few cell layers with visibly thickened cell walls. Usually, the crown is located above the level of scale leaf insertion in the bud; thus, it does not isolate scale primordia from the mature shoot tissues during initiation (Kemp, Reference Kemp1943; Lewis & Dowding, Reference Lewis and Dowding1924; Parke, Reference Parke1959; Pillai & Chacko, Reference Pillai and Chacko1978; Romberger, Reference Romberger1963; Sterling, Reference Sterling1945a, Reference Sterling1946b). By contrast, in Torreya, the crown is positioned at the level of scale leaf insertion in the bud; thus, the scale primordia develop in isolation from the mature tissues (Banasiak & Zagórska-Marek, Reference Banasiak and Zagórska-Marek2006).

Upon transition to the reproductive phase, some axillary shoots start differentiating as reproductive ovule- or pollen-bearing structures (commonly referred to as cones; for discussion on homology and development of seed cones, see Dörken & Rudall, Reference Dörken and Rudall2018; Leslie & Losada, Reference Leslie and Losada2019; Losada & Leslie, Reference Losada and Leslie2018; Matsunaga, Reference Matsunaga2026; for male cones see Schulz et al., Reference Schulz, Klaus, Knopf, Mundry, Dörken and Stützel2014; Dörken & Nimsch, Reference Dörken and Nimsch2016, Reference Dörken and Nimsch2023; Dörken, Reference Dörken2024a, Reference Dörken2024b). The meristem increases in size, broadens and changes the shape to dome-like and although the zonation pattern remains relatively stable, the divisional activity within the CMC and PZ increases (Losada & Leslie, Reference Losada and Leslie2018; Mouradov et al., Reference Mouradov, Glassick, Hamdorf, Murphy, Fowler, Marla and Teasdale1998). In the PZ, numerous bracts and ovuliferous scales are initiated, forming a complex, branched structure (Leslie & Losada, Reference Leslie and Losada2019). Upon bud breaking, the axis of a female cone starts rapid elongation that causes cone emergence; further scale differentiation and maturation result in reaching the final shape of the cone (Dörken & Rudall, Reference Dörken and Rudall2018; Jagel & Dörken, Reference Jagel and Dörken2014, Reference Jagel and Dörken2015a, Reference Jagel and Dörken2015b; Leslie & Losada, Reference Leslie and Losada2019; Losada & Leslie, Reference Losada and Leslie2018; Takaso & Tomlinson, Reference Takaso and Tomlinson1989a, Reference Takaso and Tomlinson1989b, Reference Takaso and Tomlinson1990, Reference Takaso and Tomlinson1992; Tomlinson et al., Reference Tomlinson, Takaso and Cameron1993; Tomlinson & Takaso, Reference Tomlinson and Takaso2002).

The molecular regulation of the SAM transition from vegetative to reproductive phase has been intensively studied in order to elucidate the homology and evolutionary relationships of gymnosperm and angiosperm reproductive structures (Akhter et al., Reference Akhter, Westrin, Zivi, Nordal, Kretzschmar, Delhomme, Street, Nilsson, Emanuelsson and Sundström2022; Matsunaga, Reference Matsunaga2026; Mouradov et al., Reference Mouradov, Glassick, Hamdorf, Murphy, Fowler, Marla and Teasdale1998; Song et al., Reference Song, Liu, Ma, Chen, Li, Wang, Han and Niu2025; Vázquez-Lobo et al., Reference Vázquez-Lobo, Carlsbecker, Vergara-Silva, Alvarez-Buylla, Piñero and Engström2007). The studies showed the role of C- class gene AGAMOUS in determining the identity of female (ovuliferous) structures (Matsunaga, Reference Matsunaga2026 and references therein), and the differential expression pattern of LEAFY-like (LFY-like; homologue of angiosperm LEAFY) and NEEDLY (NLY) genes that upregulate the B and C class MADS-homeobox genes (responsible in angiosperms for the identity of generative floral elements; Mouradov et al., Reference Mouradov, Glassick, Hamdorf, Murphy, Fowler, Marla and Teasdale1998; Song et al., Reference Song, Liu, Ma, Chen, Li, Wang, Han and Niu2025; Vázquez-Lobo et al., Reference Vázquez-Lobo, Carlsbecker, Vergara-Silva, Alvarez-Buylla, Piñero and Engström2007). In this context, an interesting developmental abnormality in gymnosperm cones is a resumption of vegetative growth after formation of several reproductive structures. Although a random and spontaneous process, the regrowth of vegetative shoots at the cone apex was described in several coniferous genera (Bateman et al., Reference Bateman, Hilton, Rudall, Wanntorp and De Craene2011; Dörken & Rudall, Reference Dörken and Rudall2019; Rudall et al., Reference Rudall, Hilton, Vergara-Silva and Bateman2011), and can be explained by spatiotemporal deviation in the functioning of the B and C class genes (Dörken & Rudall, Reference Dörken and Rudall2019). Observations of reverted cones could be of interest in terms of the regulation of organ positioning versus their identity. In angiosperms, vegetative organs, formed after reversion, continue the whorled phyllotaxis of floral elements regardless of a vegetative pattern (e.g., spiral; Battey & Lyndon, Reference Battey and Lyndon1990; Tooke et al., Reference Tooke, Ordidge, Chiurugwi and Battey2005), suggesting that both mechanisms are independent. In gymnosperms, although the type of vegetative and cone phyllotaxis is usually the same (Table 1), it is not known whether the expression, chirality of the pattern (for explanation of terms, see Figure 2), or the number of leaves per whorl is maintained or changes, especially given that the proportions at the SAM (i.e., the size of PZ versus primordia) change upon transition.

Figure 2.

Terminology related to spiral phyllotaxis used in the text. Drawings (a), (b), (e) – (g) after Church, Reference Church1904, modified; (b) is a mirror-image of (a).

From a quantitative perspective, the large size, prolonged activity and distinguishable cytohistological zonation of gymnosperm SAMs provide a distinct geometric and temporal context for pattern formation, affecting both pattern stability and the occurrence of transitions.

3. SAM self-maintenance regulation

Information on the genetic regulation of gymnosperm meristem functioning is fragmentary. The data come mostly from studies on somatic embryogenesis and de novo organogenesis in in vitro cultures or, alternatively, from transcriptomic analyses (Alvarez et al., Reference Alvarez, Bueno, Cañas, Avila, Cánovas and Ordás2018, Reference Alvarez, Bueno, Cuesta, Feito and Ordás2020; Bueno et al., Reference Bueno, Alvarez and Ordás2020; Hjortswang et al., Reference Hjortswang, Sundås-Larsson, Bharathan, Bozhkov, von Arnold and Vahala2002; Larsson et al., Reference Larsson, Sitbon and von Arnold2012). Recent studies showed that genetic SAM regulation relies on transcription factors belonging to the WOX (WUSCHEL-related homeobox) and KNOX (KNOTTED1-like homeobox) gene families, showing similarities to the regulatory mechanisms known in angiosperms (Alvarez et al., Reference Alvarez, Bueno, Cañas, Avila, Cánovas and Ordás2018; Bueno et al., Reference Bueno, Alvarez and Ordás2020; Hedman et al., Reference Hedman, Zhu, von Arnold and Sohlberg2013; Xu et al., Reference Xu, Hu, Chen, Tang, Chen, Chen, Cai and Xu2025). Analyses of different gymnosperm species showed the presence of the WOX genes, classified into three clades with specific expression profiles and functions. Importantly, homologues of angiosperm WUS and the gymnosperm-specific WOXX genes are possibly involved in SAM maintenance (Alvarez et al., Reference Alvarez, Bueno, Cañas, Avila, Cánovas and Ordás2018; Hedman et al., Reference Hedman, Zhu, von Arnold and Sohlberg2013; Xu et al., Reference Xu, Hu, Chen, Tang, Chen, Chen, Cai and Xu2025).

Within the KNOX family, the first gene described for gymnosperms was HBK1 (Homeobox of KNOX class) from P. abies (Sundås-Larsson et al., Reference Sundås-Larsson, Svenson, Liao and Engström1998). It shows high similarity to KN1 (KNOTTED1) from maize in terms of both the homeodomain sequence and the expression pattern. It is expressed in the central population of undifferentiated cells in the SAM but not in sites designated for new primordia formation, suggesting a control of meristem versus leaf primordia initiation similar to that in angiosperms (Hjortswang et al., Reference Hjortswang, Sundås-Larsson, Bharathan, Bozhkov, von Arnold and Vahala2002; Sundås-Larsson et al., Reference Sundås-Larsson, Svenson, Liao and Engström1998). In recent studies, more genes from class I and II of KNOX have been identified in Pinus and Picea, but their function and tissue-specific expression remain to be established (Bueno et al., Reference Bueno, Alvarez and Ordás2020).

4. Phyllotactic pattern diversity

In gymnosperms, both spiral (helical) and whorled phyllotactic patterns occur; distichy is not reported. In the majority of taxa studied so far, one species-specific phyllotactic pattern dominates, regardless of the lateral organ type (scales, assimilating leaves, cone scales, etc.; Table 1). Spiral patterns can occur in two chiral configurations depending on the course of an ontogenetic helix (winding clockwise or counterclockwise; Figure 2a,b). In gymnosperms, the frequency of both helix chiral configurations is similar and random (Banasiak & Zagórska-Marek, Reference Banasiak and Zagórska-Marek2020; Fierz, Reference Fierz2015; Gregory & Romberger, Reference Gregory and Romberger1972; Namboodiri & Beck, Reference Namboodiri and Beck1968a; Zagórska-Marek, Reference Zagórska-Marek1985).

In species with spiral phyllotaxis, the main Fibonacci pattern is usually dominant (Church, Reference Church1904; Crafts, Reference Crafts1943b; Gola & Banasiak, Reference Gola and Banasiak2016; Gregory & Romberger, Reference Gregory and Romberger1972; Jean, Reference Jean1994; Namboodiri & Beck, Reference Namboodiri and Beck1968a; Sterling, Reference Sterling1945a; Takaso & Tomlinson, Reference Takaso and Tomlinson1990; Zagórska-Marek, Reference Zagórska-Marek1985), although in some genera, for example Torreya and Cephalotaxus, the bijugy of this pattern prevails (Banasiak & Zagórska-Marek, Reference Banasiak and Zagórska-Marek2006; Kemp, Reference Kemp1943; Tomlinson & Zacharias, Reference Tomlinson and Zacharias2001; Table 1, Figure 2c,d). The other co-occurring patterns are less frequent, ranging from 3% to 5% in Abies, Picea, Pinus to ca. 20% in Larix (Banasiak & Zagórska-Marek, Reference Banasiak and Zagórska-Marek2006; Fierz, Reference Fierz2015; Zagórska-Marek, Reference Zagórska-Marek1985; Table 1). In general, several different patterns constitute a phyllotactic spectrum for a species, with additional patterns represented mostly by other monojugies (including rare phyllotactic series), multijugies (e.g., bijugies; Figure 2), and occasionally whorled patterns (e.g., decussate and tricussate).

For genera with whorled phyllotaxis, the decussate pattern is the most commonly occurring and dominant for a species (Dörken et al., Reference Dörken, Nimsch and Jagel2017; Jagel & Dörken, Reference Jagel and Dörken2014, Reference Jagel and Dörken2015a, Reference Jagel and Dörken2015b; Namboodiri & Beck, Reference Namboodiri and Beck1968b; Ruth et al., Reference Ruth, Klekowski and Stein1985; Takaso & Tomlinson, Reference Takaso and Tomlinson1989b; Table 1). Some genera are generalised as having decussate or tricussate phyllotaxis, though the frequencies of the two patterns are not given (e.g., in Juniperus, subfamilies Cupressoideae, Callitroideae; Jagel & Dörken, Reference Jagel and Dörken2015a, Reference Jagel and Dörken2015b). In the Callitroideae, the tricussate pattern is more common than in the Cupressoideae, and this is the typical phyllotaxis of Fitzroya, Callitris, Actinostrobus (at least in cone bracts; Jagel & Dörken, Reference Jagel and Dörken2015b; Takaso & Tomlinson, Reference Takaso and Tomlinson1989b). Two genera, Neocallitropsis and Actinostrobus, are characterised by a tetracussate arrangement of leaves on adult vegetative shoots and of scales in cones (Jagel & Dörken, Reference Jagel and Dörken2015b; Table 1).

5. Structure of the primary vascular system

The primary vascular system of gymnosperms is composed of discrete axial vascular bundles and regularly diverging leaf traces (i.e., vascular strands supplying lateral organs). Each axial vascular bundle, together with related leaf traces, forms a vascular sympodium (Evert, Reference Evert2007; Namboodiri & Beck, Reference Namboodiri and Beck1968a, Reference Namboodiri and Beck1968b). The close relation between superficial leaf arrangement (phyllotaxis) and the internal vascular supply of leaves is not surprising, as both structures develop within the PZ of the SAM and, in angiosperms, are related to a shared mechanism of polar auxin transport (PAT) (Banasiak & Gola, Reference Banasiak and Gola2023; Reinhardt et al., Reference Reinhardt, Pesce, Stieger, Mandel, Baltensperger, Bennett, Traas, Friml and Kuhlemeier2003). Procambium, the vascular tissue progenitor, develops in the inner region of the PZ as strands of slightly elongated cells. Each procambial strand divides, and one part departs, forming a leaf trace (initiated in the outer region of the PZ), whereas the other part of a strand continues as the axial vascular bundle (Esau, Reference Esau1965; Evert, Reference Evert2007).

Interestingly, in some genera, for example Taxus, Pseudotsuga, Sequoia, the presence of procambial leaf traces was observed before the initiation of the corresponding leaf primordia (Gunckel & Wetmore, Reference Gunckel and Wetmore1946a; Romberger, Reference Romberger1963; Sterling, Reference Sterling1945b, Reference Sterling1947). In addition, differentiated vascular elements could be present in the basal parts of such leaf traces (Sterling, Reference Sterling1946b). The opposite tendency in leaf trace differentiation was observed in Abies, where the youngest five or six initiated leaf primordia in the terminal bud were devoid of the procambial strands, which started developing later, during embryonic shoot elongation (Banasiak & Gola, Reference Banasiak and Gola2023; Parke, Reference Parke1963).

The primary vascular system in species with helical phyllotaxis shows high regularity: the number of sympodia belongs to the same phyllotactic series as the superficial leaf pattern, and their orientation agrees with the chirality of the same number of leaf parastichies in a given phyllotactic series, but departing leaf traces show the opposite chirality (Namboodiri & Beck, Reference Namboodiri and Beck1968a; Romberger et al., Reference Romberger, Hejnowicz and Hill1993). Only a single leaf trace supplies a leaf primordium, with the exception of Ginkgo (Gunckel & Wetmore, Reference Gunckel and Wetmore1946b) and Ephedra (Dörken, Reference Dörken2012), where two leaf traces from neighbouring axial bundles supply a leaf. However, as there are no lateral connections between axial bundles or leaf traces (even in the case of Ginkgo and Ephedra), each sympodium maintains its discreteness (Namboodiri & Beck, Reference Namboodiri and Beck1968a).

The primary vascular system in species with whorled phyllotaxis is more variable. It can resemble that in gymnosperms with helical phyllotaxis (the number of sympodia corresponding to orthostichy numbers; single leaf traces supplying leaf primordia), but the chirality of leaf traces can be randomised. Importantly, in the evolutionarily advanced Cupressaceae, two leaf traces diverge from two neighbouring axial bundles toward the leaf (as in the case of Ginkgo and Ephedra), but they fuse, forming connections between axial bundles and closing the primary vascular system (Namboodiri & Beck, Reference Namboodiri and Beck1968b).

Interestingly, phyllotaxis and the primary vascular system are related to another internal system, present in some gymnosperm genera, that is axial cortical resin ducts. Their number is a member of the same phyllotactic series, always the one preceding the number of sympodia in the sequence and having an opposite orientation (Zagórska-Marek & Banasiak, Reference Zagórska-Marek and Banasiak2000). The underlying cause of this interrelationship remains unknown, although spatiotemporal dependencies could suggest inductive interactions between these systems.

The structure of the primary vascular system was studied in search of the anatomical basis of the above-mentioned homo- and antidromic phyllotactic relations between the main and lateral axes. Surprisingly, regardless of the lack of correlations and the randomised course of the ontogenetic helix in Picea and Abies, their sympodial structure shows almost complete homodromy. The chiral orientation of sympodia in the main and lateral axes remains the same. Interestingly, the same correlation was observed in Cedrus, in which leaf phyllotactic antidromy occurred. Importantly, in the studied genera, if the chirality of sympodia changed, the orientation in lateral axes followed this change accordingly. This suggests that leaf phyllotaxis adjusts to the internal vascular organisation of the shoot (Banasiak & Zagórska-Marek, Reference Banasiak and Zagórska-Marek2020).

These results imply that the primary vascular system can act as a structural constraint that cooperates with the SAM, affecting pattern stability, chirality maintenance and continuation along one axis. Understanding these interactions and underlying mechanisms requires, however, the creation and implementation of markers – not yet available for gymnosperms – for tracing subsequent stages of vascular system differentiation.

6. Phyllotaxis regulation

The auxin-dependent mechanism of phyllotaxis regulation in gymnosperms remains poorly studied and presents a great challenge due to a lack of model systems, huge genomes of gymnosperms and limited availability of genetic tools and molecular markers. In addition, the longevity of species, seasonal growth and methodological problems with the analysis of auxin transport result in limited knowledge of PAT and its role in phyllotaxis regulation in gymnosperms. Some information can, however, be inferred from the genomic and phylogenetic analyses.

The main sites of auxin biosynthesis are the SAM and young leaves, as in angiosperm plants. In addition, YUCCA genes, involved in the IPA-dependent main auxin biosynthetic pathway, have been identified in gymnosperms (Vijayanathan et al., Reference Vijayanathan, Faryad, Abeywickrama, Christensen and Jakobsen Neilson2025).

PAT has been detected in gymnosperms and, like in angiosperms, it involves three families of evolutionarily conserved proteins, PIN (PINFORMED), AUX/LAX (AUXIN1/LIKE-AUX1) and ABC (ATP-BINDING CASSETTE TRANSPORTER; Bennett, Reference Bennett2015; Carrillo-Carrasco et al., Reference Carrillo-Carrasco, Hernandez-Garcia, Mutte and Weijers2023; Salazar et al., Reference Salazar, Pollmann, Morales-Quintana, Herrera, Caparrós-Ruiz and Ramos2019).

In P. abies, the PaPIN1 protein has been identified and characterised, and despite differences in structure, shows similarities to AtPIN1 in the expression pattern during embryogenesis. It has strong expression in the protoderm during cotyledons initiation and then in cotyledon tips and developing procambium (Palovaara et al., Reference Palovaara, Hallberg, Stasolla, Luit and Hakman2010). Experiments also show that the inhibition of PAT by NPA (N-1-naphthylphthalamic acid) in P. abies leads to embryonic SAM impediment, fusion of cotyledons and changes in procambial strands (Hakman et al., Reference Hakman, Hallberg and Palovaraa2009; Holloway et al., Reference Holloway, Rozada and Bray2018). Interestingly, NPA treatment causes overexpression of PaPIN1 and PaWOX2, whose activity in embryos is PAT-related, suggesting a compensation mechanism (Hassani et al., Reference Hassani, Trontin, Raschke, Zoglauer and Rupps2022). The effects of PAT inhibition resemble those in angiosperms and can imply similar regulation of embryonic organ development (Hakman et al., Reference Hakman, Hallberg and Palovaraa2009). Despite the variable number of cotyledons in gymnosperms, the PAT-dependent mechanism of their initiation seems to be evolutionarily conserved in both groups of plants. Other homologues of PIN proteins, PtPINE, PtPING and PtPINH, were identified in P. taeda (Bennett, Reference Bennett2015; Palovaara et al., Reference Palovaara, Hallberg, Stasolla, Luit and Hakman2010; Zhang et al., Reference Zhang, Rodriguez, Li, Zhang and Friml2020). Used in heterologous complementing systems in Arabidopsis pin1 mutant, PtPINE and PtPINH can rescue the phenotype, leading to organ formation and inflorescence development (Zhang et al., Reference Zhang, Rodriguez, Li, Zhang and Friml2020).

Less information is available about the two remaining groups of auxin plasma membrane transporters. The AUX/LAX family in gymnosperms is represented by a lower number of proteins compared to angiosperms; for example in Pinus radiata, three transporters have been described: AUX1–1, AUX1–2 and AUX1–3, which participate in auxin maxima formation necessary for vascular tissue formation (Salazar et al., Reference Salazar, Pollmann, Morales-Quintana, Herrera, Caparrós-Ruiz and Ramos2019). The ABC transporters (group B) were identified in Pinus and Picea, where they are involved in cambium activity and secondary growth regulation (Dobrowolska et al., Reference Dobrowolska, Businge, Abreu, Moritz and Egertsdotter2017; Salazar et al., Reference Salazar, Pollmann, Morales-Quintana, Herrera, Caparrós-Ruiz and Ramos2019).

The auxin-dependent regulatory mechanism is studied using fusion proteins with GFP, GUS or luciferase. They are implemented in heterologous systems where gymnosperm proteins (e.g., from Pinus taeda) are expressed in model plants (mostly Arabidopsis), during somatic embryogenesis, and in callus tissues (Feng et al., Reference Feng, Song and Xu2026; Höfig et al., Reference Höfig, Moyle, Putterill and Walter2003; Lee et al., Reference Lee, Moon and Park2014; Taniguchi et al., Reference Taniguchi, Ohmiya, Kurita and Kondo2004; Zhao et al., Reference Zhao, Zhang, Zhao and Niu2024). Although these systems are utilised to study different processes, the tool can potentially be modified to analyse SAM and phyllotaxis regulation. However, it would also require developing an experimental setup to study auxin-related processes and markers in mature plants, as so far mostly embryos have been analysed.

Developing stable and widely applicable transformation systems for gymnosperms remains a major challenge because of their complex biology, long life cycle and low efficiency of tissue regeneration in culture. The other problems are low efficiency and stability of transformation (often related to the particular genotype; thus, no single universal protocol is available for all species), the lack of a gymnosperm model system and gymnosperm biochemical specificity. Despite this, the first reports of stable gymnosperm transformation came from the end of the 20th century, when transgenic Larix plants were obtained (Huang et al., Reference Huang, Diner and Karnosky1991; Levée et al., Reference Levée, Lelu, Jouanin, Cornu and Pilate1997). Since then, several gymnosperm species (e.g., Pinus taeda, P. sylvestris, P. radiata, P. abies, Pseudotsuga menziesii) have been successfully transformed. Gymnosperm transformations as well as gene editing by the CRISP/Cas9 system have been successfully used to analyse different developmental and physiological processes, specifically gene functions and the regulatory mechanisms of growth and reproduction (Rodríguez et al., Reference Rodríguez, Ordás and Alvarez2022; Zhao et al., Reference Zhao, Zhang, Zhao and Niu2024). Thus, gymnosperms offer a system in which conserved molecular machinery, acting within spatiotemporal and anatomical constraints, can reveal principles of organ patterning regulation that are unavailable in short-lived angiosperm models.

7. Conclusions and perspectives

The classical auxin-dependent model, developed mostly based on Arabidopsis, seems to be universal in its principles across seed plants. However, it has become generally accepted that the model requires the incorporation of additional factors, not all of which are yet known. The extension of research into gymnosperms – the other, distantly related group of plants – will possibly enable analysis of the emergence and evolution of phyllotaxis regulatory mechanisms from a different perspective. In this context, gymnosperms are promising, although forgotten, models for understanding phyllotaxis regulation.

Gymnosperm SAMs are structurally similar to those in angiosperms but devoid of tunica layers (whole structure is similar to the angiosperm corpus); however, due to limited periclinal divisions in some species, a pseudotunica can be formed that resembles the L1 layer of the angiosperm meristem. This structural similarity could be translated into a similar regulatory mechanism of leaf patterning, assuming the presence of homologue PIN proteins and PAT in gymnosperms.

Relatively huge SAMs and the availability of numerous organ primordia for organogenesis manipulation are beneficial for implementing different experimental setups for analyses of the regulation of leaf positioning, both empirical and computational. Furthermore, well-described phyllotaxis diversity and its ontogenetic changes, together with the well-recognised structure and development of internal systems, constitute huge datasets for future analyses.

Research on gymnosperm phyllotaxis can reach a new level, as genomes of some coniferous species have now been sequenced (at least partially), and methods of transformations and gene editing, as well as techniques for visualising gene/protein expression, have become available. Although developed to analyse other developmental or cellular processes, this knowledge can be advantageous in the search of the molecular background of organ patterning regulation in gymnosperms.