Development, structure, and secretion of leaf colleters in Clusia criuva Cambess. subsp. criuva (Clusiaceae)

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Introduction
Throughout the evolution of terrestrial plants, protection strategies have been incorporated into the plant body that allow for greater competition and resistance to environmental adversities, thus ensuring the survival of plants in different environments (Paiva & Machado 2006;Paiva 2016;Prado & Demarco 2018).One of these strategies involves chemical defense through compounds produced by secretory structures (Prado & Demarco 2018).Plant secretions are often associated with defense strategies (Paiva 2016).They are constituted with different primary and/ or secondary metabolites.The chemical predominance of certain compounds in the secretion may suggest specificity in the activity of the secretory cells and also on the ecological role played by such secretion (Castro & Machado 2006;Ascensão 2007;Paiva 2016).
Acta Botanica Brasilica, 2022, 36: e2021abb0103 Colleters are external structures that produce a sticky secretion composed of mucilage, resins, or a mixture of both (Fahn 1979;Thomas 1991).The colleter secretion present on the surface of vegetative and reproductive organs, protects developing meristems and organs against desiccation (Thomas 1991), acts as an insect repellent (Smith 1963;Curtis & Lersten 1974) or herbivore and pathogen deterrent, and provides inhibition of fungi and bacterial growth through its antimicrobial properties (Castro & Demarco 2008;Calvo et al. 2010;Cardoso-Gustavson et al. 2014).The colleters have an early development and usually complete their differentiation and even their senescence long before the final development of the plant organ that surrounds them (Canaveze 2012).
Colleters are widely distributed among the angiosperms and were reported in about 60 families (Thomas 1991), including Clusiaceae (Malpighiales), a family known worldwide for the economic importance of its secretions (Judd et al. 2009).However, the structural and biological aspects of colleters are poorly investigated within the family being restricted to Garcinia and Clusia.Filiform colleters occur in the flowers of Garcinia brasiliensis (Leal et al. 2012); lachrymiform colleters (cone shaped) were found on the leaf axes of Clusia fluminensis and C. lanceolata (Silva et al. 2019) and on the petiole of C. grandiflora (Machado & Emmeric 1981).Recently, standard type colleters were found at the base of the petiole of C. burchellii (Teixeira et al. 2021).
Clusia is one of the largest genera of the Clusiaceae (300 -400 spp.) with 68 species found in different Brazilian biomes (Lüttge 2007;BFG et al. 2015;Bittrich et al. 2015).During field expeditions in a fragment of Atlantic Forest located in the State of Espírito Santo (Brazil), the observation of copious secretions covering the apical meristems and young leaves of Clusia criuva subsp.criuva led us to hypothesize that such exudates were produced by colleters.In the present study we aim to confirm the structure identity responsible for the exudation of the secretion observed in the field.Additionally, we discussed the chemical composition of the secretion throughout the leaf development in a way to contribute to the understanding of the morphofunctionality of such leaf glands present in Clusiaceae.

Collection and sampling location
Samples of the (i) shoot apical meristem with leaf primordia and the base of (ii) young (first and second nodes), (iii) completely expanded (third and fourth nodes) and (iv) senescent leaves (sixth node on) were collected (Fig. 1A -D).The samples were collected from three individuals of Clusia criuva Cambess.subsp.criuva in the Parque Estadual da Pedra Azul (20°24' S, 41°01' W), in the municipality of Domingos Martins, located in the state of Espírito Santo, Brazil (Fig. 1E).The area is an Upper Montane Rain Forest with rupestrian vegetation (Fig. 1F) which belongs to the Atlantic Forest Domain.Female (Fig. 1G) and male (Fig. 1H) individuals were collected in a transition region from forest to rupestrian area, growing on a rocky outcrop in full sun (Fig. 1I).Reproductive branches were dried and deposited in the collection of the herbarium at the Instituto Federal Goiano, campus Rio Verde (IFRV): Dalvi VC 111,Dalvi VC 112 and Dalvi VC 113.The species identification was confirmed by Dr. José Elvino do Nascimento Júnior.

Light microscopy
In the field, the samples were fixed in FAA (formalin, acetic acid, and 70 % ethanol 1:1:18 v/v) (Johansen 1940) for structural characterization, as well as in neutral-buffered formalin solution (phosphate buffer: formalin, 9:1 v/v) (Lillie 1965) for histochemical tests.After 48 h, all samples were subjected to dehydration in an ethanol series until their storage in 70% ethyl alcohol.
Samples fixed in neutral-buffered formaldehyde solution were included in methacrylate resin, as described above, and subjected to histochemical tests including: periodic acid and Schiff's reagent (McManus 1948); ruthenium red (Johansen 1940); potassium dichromate (Gabe 1968) and ferric chloride (Johansen 1940); Sudan III (Pearse 1985); and Coomassie brilliant blue (Fisher 1968) for detection of total polysaccharides, pectins/mucilage, phenolic compounds, total lipids, and proteins, respectively.The sections with no reagent (control) were also observed under a light microscope.Material analysis and photographic documentation were performed using an Olympus (model BX61) light microscope equipped with an image capture system, a DP-72 camera.

Scanning Electron Microscopy
For scanning electron microscopy, leaf samples of all four developmental stages were fixed in Karnovsky's fixative (0.1 M sodium phosphate buffer, pH 7.2) (Karnovsky 1965) for 48 h.After dehydration in an ethanol series, the specimens were critical point-dried using CO 2 (Autosamdri®, 815, Séries A) (Bozzola & Russel 1992), mounted on stubs with double-sided adhesive tape, and sputter-coated with gold (Denton Vacuum, Desk V).Photographs were taken using a scanning electron microscope (Jeol, JSM -6610), equipped with Energy-Dispersive X-Ray Spectroscopy (EDS) and NSS Spectral Imaging (Thermo Scientific, USA).

Variations in the morphology and secretory activity of the colleters
The colleters were located on the adaxial surface of the leaves, in the region of insertion with the stem, and they presented different coloration according to the leaf developmental stages (Fig. 2).We observed a large accumulation of translucent secretion (Fig. 2A-C) covering the leaf primordia.The young leaves displayed three to four rows of colleters with a whitish color and a brownish apical portion (Fig. 2D).In these two stages we observed asynchrony in the development of the colleters and the beginning of senescence was noticeable by the wilting  of cells at the apical portion (Fig. 2E).Intense production and release of secretion via cuticular rupture (Fig. 2F-G) was observed.
In the adult leaves, the brown color of the colleters was intensified and a reduction in the amount of secretion was observed both in the field (Fig. 2H) and with SEM (Fig. 2I).In the apical portion of the colleter, a constriction was noted that separates the brownish apex, where wilting and cell disruptions were common, from the lower region (Fig. 2I).
Finally, colleters acquired a blackish color (Fig. 2J), coinciding with the senescence of the leaves.In this phase little or no secretion was observed and the colleters were withered (Fig. 2K).Cellular disruptions, especially in the secretory portion, were common (Fig. 2L).

Development and histology of colleters
Colleters were formed during the first stage of leaf development (Fig. 3).In early development, most cells of the protoderm elongated (Fig. 3A) and successive anticlinal divisions resulted in a projection formed by the multiseriate epidermis (Fig. 3A).The newly divided cells presented dense cytoplasm, voluminous nuclei and nucleoli, and high nucleus/cytoplasm ratio (Fig. 3B).The subepidermal cells then divided and formed the central axis made up of parenchymal cells (Fig. 3A, D), and at this stage, the vacuolization process was observed throughout the cytoplasm of the parenchymal cells (Fig. 3C).Divisions were intensified, culminating in the formation of colleters (Fig. 3D).
Colleters were sessile and non-vascularized with a truncated or tapered apex (Fig. 3D-E).They presented a multicellular head formed by a central axis of parenchymal cells and a secretory epidermis (Fig. 3D-F) coated with a thick cuticle (Fig. 3G), following into the standard-type colleters.The secretory epidermis had commonly more than two layers of columnar juxtaposed cells which tended to palisade (Fig. 3E).
In adult leaves, the degradation of cells in the apical portion was accentuated (Fig. 3H).In senescent leaves, the secretory epidermis of most colleters was completely degraded with collapsed and/or ruptured cells (Fig. 3I-J).Although senescent, Clusia criuva subsp.criuva colleters did not come into abscission.

Histochemical characterization
Histochemical tests showed polysaccharides as the main components of the secretion produced by Clusia criuva subsp.criuva.Total polysaccharides (Fig. 4A-C) and pectins/mucilage (Fig. 4D-F) were observed in the exudates of the colleters at all leaf development stages.In the primordia, young, and adult leaves, accumulation of these compounds was observed in the cytoplasm of secretory cells (Fig. 4A, B, D, E), in the subcuticular space (Fig. 4B, E), and in extravasated secretion (Fig. 4A, C, F).
Proteins were observed in the exudate of the colleters from the leaf primordia (Fig. 4G) through to the senescent leaves (Fig. 4I).However, the accumulation of proteins in secretory cells was evident only in colleters from leaf primordia and young leaves (Fig. 4G).Lipid droplets were observed within the secretory cells as well as in the cells from the central axis in the young leaves.In adult and senescent leaves, lipids were detected only in secretory epidermal cells (Fig. 4H), and structural lipids impregnated in cell walls in cells of the central axis (Fig. 4H).
Phenolic compounds were registered in the colleters present in all leaf development stages.However, in leaf primordia and young leaves, these compounds were restricted to the apical portion of the colleters (Fig. 4J), whereas in adult and senescent leaves, phenolics were observed throughout the entire structure of the colleter, particularly in the secretory cells of the epidermis (Fig. 4K, L).

Discussion
The abundant secretion observed in the field in the leaf primordia and young leaves of Clusia criuva subsp.criuva, the distribution as well as the structural and chemical nature of the secretion confirm our hypothesis for the presence of colleters on the leaves of this species.The colleters of C. criuva subsp.criuva are derived from the protoderm and the ground meristem and are classified as emergencies according to Evert (2006).In Ilex species, González (1998) reported that a group of protodermal cells undergoes radial enlargement and anticlinal division and some subepidermal cells divide to form a protuberance.Thus, the anticlinal division gives rise to the secretory epidermis and the subepidermal cells produce the parenchymal axis of the colleter, and the same pattern has been observed here.Other studies report that the most common occurrence is that of colleters forming only by the protoderm from periclinal and anticlinal divisions, without no involvement of the ground meristem (Paiva & Machado 2006;Paiva 2009;Paiva & Martins 2011;Rocha et al. 2011).As pointed out by our results, the involvement of the protoderm and the ground meristem in the process for colleter was reported in C. fluminensis, C. lanceolata (Machado & Emmerich 1981;Silva et al. 2019) and C. burchellii (Teixeira et al. 2021), which suggests that it may be a conservative characteristic in the group, although studies on colleters in the genus are still scarce.
The standard type colleters present in C. criuva subsp.criuva is constituted by a central parenchymal axis surrounded by a layer of palisade-secreting epidermal cells, as previously reported by Lersten (1974).The standard type colleter also was found in C. grandiflora (Machado & Emmerich 1981) and C. burchelli (Teixeira et al. 2021).In C. fluminensis and C. lanceolata, lachrymiform cone-shaped colleters were described (Silva et al. 2019).However, we understand that the lachrymiform refers to the external morphology of the colleter, which is anatomically organized as the standard type, according to Lersten (1974).Studies on the morphological differences of colleters can be important to offer contributions to taxonomy studies and to establish phylogenetic relationships within the Clusiaceae.Our results clearly show cuticle ruptures in the apical portion of the colleters, which is a common way to secrete substances by colleters (Paiva & Machado 2006;Silva et al. 2017).
The chemical composition of the colleter exudates in C. criuva subsp.criuva is diverse as it is composed of a mixture of hydrophilic and lipophilic compounds, that is, polysaccharides, proteins, lipids, and phenolic compounds.However, variations in the composition of the secretion were observed depending on the leaf development stage.Mucilage consisting mainly of polysaccharide polymers with high molecular weight (Fahn 1988) together with pectin may act as a water absorber due to its hygroscopic properties (Hall 1981;Nobel et al. 1992), which may protect meristem and young leaves of C. criuva subsp.criuva against desiccation.This corroborates the primary function of colleters in producing secretions that protect young organs during their development (Lersten & Horner 1968;Lersten 1974;Fahn 1979) and/or that promote the lubrication of the stem meristem, minimizing friction of the developing leaf tissues and preventing dehydration (Fahn 1979;Thomas 1991;Mayer et al. 2011;Silva et al. 2012;Mayer et al. 2013).Proteins are involved in the defense against fungal activity and attack of pathogens (Klein et al. 2004;Miguel et al. 2006) and are commonly reported in the secretion of colleters (González & Tarragó 2009;Coelho et al. 2013;Dalvi et al. 2014;Lacchia et al. 2016;Pinheiro et al. 2019).
The phenolic compound production was intensified, in colleters, of adult and senescent leaves of C. criuva subsp.criuva.Phenolics have been reported for other species of Clusia both in the secretion present in the meristem and in the adult leaves (Silva et al. 2019).According to Coelho et al. (2013), phenolic compounds and lipids are the final products of the secretion of colleters in a senescence stage.The cuticle wrinkling, the formation of a constriction at the base of the colleters and cell disruption also indicate the senescence of the colleters (Pinheiro et al. 2019), which were also shown here for C. criuva subsp.criuva colleters even they did not come into abscission.
The color of colleters changes in the field during the development of C. criuva subsp.criuva leaf, as reported for C. burchellii (Teixeira et al. 2021) and species from other families, including Apocynaceae (Thomas & Dave 1989;Appezzato-da-Glória & Estelita 2000;Rio et al. 2002), Gentianaceae (Dalvi et al. 2013;2014), and Rubiaceae (Lersten 1974;Miguel et al. 2006;2009;Vitarelli & Santos 2009;Klein et al. 2010;Pinheiro et al. 2019).These authors generally associate changes in color with the presence of phenolic compounds, which was confirmed in colleters of C. criuva var.criuva.The accumulation of phenolic compounds in colleters is also related to a change in the function of these glands, being produced in a second stage of the secretory phase (Ribeiro et al. 2017), which would act to avoid predation (Castro & Demarco 2008).However, the position of colleters in this species and others would not preclude the predation of leaves by herbivores.
In summary, we report here the occurrence of standard-type colleters in C. criuva subsp.criuva as well as the morphoanatomical variations of the structure and chemical composition of the exudate throughout the leaf development of this species.Our results contribute to a better understanding of the morphofunctionality of colleters in Clusiaceae, structures that are scarcely studied in this group of plants.Comparative studies with a larger number of Clusiaceae species are also needed in order to understand the evolution of colleter for this family and their correlation with the biomes where such species occur.

Figure 1 .
Figure 1.Map with the geographic distribution of the Atlantic Forest in Brazil, collection site of Clusia criuva Cambess.subsp.criuva and sampled material.A-D.Leaves at different developmental stages.A. Stem apical meristem with leaf primordia (arrow).B. Young leaf.C. Adult (completely expanded) leaf.D. Senescent leaves.E. Map for the sampling area, Domingos Martins, State of Espírito Santo, Brazil.F. Collection site (arrow) near the Parque Estadual da Pedra Azul.G. Female flower.H. Male flower.I. Details of individual (arrow) growing in a rocky outcrop area.

Figure 4 .
Figure 4. Histochemical tests of Clusia criuva Cambess.subsp.criuva.A-C.Reaction with PAS showing the presence of polysaccharides.D-F.Presence of pectin confirmed by the red ruthenium test.G, I. Reaction with Coomassie brilliant blue showing proteins in the cytoplasm and extravasated secretion.H. Lipid droplets (black arrow) and impregnation of lipids in the cells of the central axis (white arrow), evidenced by Sudan III.J-L.Ferric chloride test demonstrates the increase in the amounts of phenolic compounds in the colleters during leaf development.Asterisks indicate extravasated secretion.Bars: A, D, G, J = 500 µm; B, C, E, F, I, K, L = 100 µm; H = 20 µm.