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

ABSTRACT

Colleters produce a secretion composed of hydrophilic and/or lipophilic substances which lubricates and protects the shoot apical meristem against biotic and abiotic agents. Little attention has been given to these structures in Clusiaceae. In the present study, the structure and development of the leaf colleters of Clusia criuva subsp. criuva were described and variations in the exudate composition of the colleters at different stages of leaf development were identified. The samples were collected and processed according to techniques for light and scanning electron microscopy. Colleters are of the standard type and not vascularized, and during leaf development, changes in color, structure, and secretion abundance were observed. Asynchrony in the development was noticeable in the leaf primordia and young leaves, from colleters in early formation to those in early senescence. In early phases there was an abundance of polysaccharide, lipid, and protein secretions, whereas adult and senescent leaves revealed an accumulation of phenolic compounds and cell degradation. The secretion was released by the rupture of the cuticle. The structural changes and secretion composition during leaf development emphasize the role of colleters in protecting meristems and developing organs.

Keywords:
exudate; glands; histochemistry; Malpighiales; morphoanatomy

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 2006Paiva EAS, Machado SR. 2006. Colleters in Caryocar brasiliensis(Caryocaraceae), ontogenesis, ultrastructure and secretion. Brazilian Journal of Biology 66: 301-308.; Paiva 2016Paiva EAS. 2016. How do secretory products cross the plant cell wall to be released? A new hypothesis involving cyclic mechanical actions of the protoplast. Annals of Botany 117: 533-540.; Prado & Demarco 2018Prado E, Demarco D. 2018. Laticifers and secretory ducts: similarities and differences. In: Hufnagel L (ed.) Ecosystem services and global ecology. London, IntechOpen. p. 103-123.). One of these strategies involves chemical defense through compounds produced by secretory structures (Prado & Demarco 2018Prado E, Demarco D. 2018. Laticifers and secretory ducts: similarities and differences. In: Hufnagel L (ed.) Ecosystem services and global ecology. London, IntechOpen. p. 103-123.). Plant secretions are often associated with defense strategies (Paiva 2016Paiva EAS. 2016. How do secretory products cross the plant cell wall to be released? A new hypothesis involving cyclic mechanical actions of the protoplast. Annals of Botany 117: 533-540.). 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 2006Castro MM, Machado SR. 2006. Células e tecidos secretores. In: Appezzato-da-Glória B, Carmello-Guerreiro SM (eds.) Anatomia vegetal. 2nd. edn. Viçosa, Universidade Federal de Viçosa. p. 179-2013.; Ascensão 2007Ascensão L. 2007. Estruturas secretoras em plantas. Uma abordagem morfo-anatômica. In: Figueiredo AC, Barroso JG, Pedro LG. (eds.) Potencialidades e aplicações das plantas aromáticas e medicinais. Curso Teórico-Prático. 3. ed. Lisboa: Faculdade de Ciências da Universidade de Lisboa/Centro de Biotecnologia Vegetal. p. 19-28.; Paiva 2016Paiva EAS. 2016. How do secretory products cross the plant cell wall to be released? A new hypothesis involving cyclic mechanical actions of the protoplast. Annals of Botany 117: 533-540.).

Colleters are external structures that produce a sticky secretion composed of mucilage, resins, or a mixture of both (Fahn 1979Fahn A. 1979. Secretory Tissues in Plants. London, Academic Press.; Thomas 1991Thomas V. 1991. Structural, functional and phylogenetic aspects of the colleter. Annals of Botany 68: 287-305.). The colleter secretion present on the surface of vegetative and reproductive organs, protects developing meristems and organs against desiccation (Thomas 1991Thomas V. 1991. Structural, functional and phylogenetic aspects of the colleter. Annals of Botany 68: 287-305.), acts as an insect repellent (Smith 1963Smith RH. 1963. Preferential attack by Dendroctonus terebrans on Pinus elliotti. Journal of Economic Entomology 56: 817-819.; Curtis & Lersten 1974Curtis JD, Lersten NR. 1974. Morphology, seasonal variation, and function of resin glands on buds and leaves of Populus deltoides (Salicaceae). Botany 61: 835-845.) or herbivore and pathogen deterrent, and provides inhibition of fungi and bacterial growth through its antimicrobial properties (Castro & Demarco 2008Castro MM, Demarco D. 2008. Phenolic compounds produced by secretory structures in plants: a brief review. Natural Product Communications 3: 1273-1284.; Calvo et al. 2010Calvo TR, Demarco D, Santos FV, et al. 2010. Phenolic compounds in leaves of Alchornea triplinervia: anatomical localization, mutagenicity, and antibacterial activity. Natural Product Communication 5: 1225-1232. ; Cardoso-Gustavson et al. 2014Cardoso-Gustavson P, Campbell LM, Viveiros-Mazzoni S, Barros F. 2014. Floral colleters in Pleurothallidinae (Epidendroideae: Orchidaceae). American Journal of Botany 101: 587-597.). 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 2012Canaveze Y. 2012. Estrutura, origem e desenvolvimento de laticíferos e coléteres em plantas de Tabernaemontana catharinensis a.dc. (Rauvolfioideae, Apocynaceae) em diferentes fases do desenvolvimento vegetativo. MSc Thesis, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Botucatu, Brasil.).

Colleters are widely distributed among the angiosperms and were reported in about 60 families (Thomas 1991Thomas V. 1991. Structural, functional and phylogenetic aspects of the colleter. Annals of Botany 68: 287-305.), including Clusiaceae (Malpighiales), a family known worldwide for the economic importance of its secretions (Judd et al. 2009Judd WS, Campbell CS, Kellogg EA, Stevens PF, Donoghue MJ. 2009. Sistemática Vegetal - um enfoque filogenético 3rd. edn. Porto Alegre, Artmed.). 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. 2012Leal DO, Malafaia C, Cesar R, et al. 2012. Floral structure of Garcinia brasiliensis in relation to flower biology and evolution. International Journal of Plant Sciences 173: 172-83.); lachrymiform colleters (cone shaped) were found on the leaf axes of Clusia fluminensis and C. lanceolata (Silva et al. 2019Silva KMM, Luna BN, Joffily A, Paiva SR, Barros CF. 2019. Revealing the development of secretory structures in the leaves of Clusia fluminensis and Clusia lanceolata (Clusiaceae). Flora 256: 69-78.) and on the petiole of C. grandiflora (Machado & Emmeric 1981Machado MMP, Emmerich M. 1981. Presença de coléteres em Clusia lanceolata Cambess. Boletim do Museu Nacional de Botânica 59: 1-7.). Recently, standard type colleters were found at the base of the petiole of C. burchellii (Teixeira et al. 2021Teixeira RS, Rocha DI, Dalvi V. 2021. Leaf colleters in Clusia burchellii Engl.: Structural and ultrastructural features of a little-known gland in Clusiaceae. Flora 280: 151834.).

Clusia is one of the largest genera of the Clusiaceae (300 - 400 spp.) with 68 species found in different Brazilian biomes (Lüttge 2007Lüttge U. 2007. Clusia - A woody Neotropical genus of remarkable plasticity and diversity. Ecological studies, vol. 194. New York, Springer. ; BFG 2015BFG - The Brazil Flora Group. 2015. Growing knowledge: an overview of seed plant diversity in Brazil. Rodriguésia 66: 1085-1113.; Bittrich et al. 2015Bittrich V, Trad RJ, Cabral FN, Nascimento-Jr JE, Souza VC. 2015. Clusiaceae in lista de espécies da flora do Brasil. Jardim Botânico do Rio de Janeiro. http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB28292. 03 Jan. 2020.
http://floradobrasil.jbrj.gov.br/jabot/f...
). 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.

Material and methods

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°24ʹ S, 41°01ʹ 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.

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.

Light microscopy

In the field, the samples were fixed in FAA (formalin, acetic acid, and 70 % ethanol 1:1:18 v/v) (Johansen 1940Johansen DA. 1940. Plant microtechnique. New York, Mc GrawHill.) for structural characterization, as well as in neutral-buffered formalin solution (phosphate buffer: formalin, 9:1 v/v) (Lillie 1965Lillie RD. 1965. Histopathologic technic and practical histochemistry. 3rd. edn. New York, McGraw-Hill.) for histochemical tests. After 48 h, all samples were subjected to dehydration in an ethanol series until their storage in 70% ethyl alcohol.

Subsequently, the samples were embedded in methacrylate resin (Historesin Leica; Leica Microsystems, Heidelberg, Germany), transversely and longitudinally sectioned using a rotary microtome (1508R; Logen Scientific). Sections at 5 µm thickness were stained with toluidine blue (pH 4.6) (O'Brien et al. 1964O’Brien TP, Feder N, Mccully ME. 1964. Polychromatic staining of plant cell walls by toluidine blue O. Protoplasma 59: 368-373.), mounted on slides with synthetic resin (Permount; Fisher Scientific, NJ, USA) and anatomically characterized.

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 1948McManus JFA. 1948. Histological and histochemical uses of periodic acid. Stain Technol. 23: 99-108.); ruthenium red (Johansen 1940Johansen DA. 1940. Plant microtechnique. New York, Mc GrawHill.); potassium dichromate (Gabe 1968Gabe M. 1968. Techniques histologiques. Paris, Masson and Cie. ) and ferric chloride (Johansen 1940Johansen DA. 1940. Plant microtechnique. New York, Mc GrawHill.); Sudan III (Pearse 1985Pearse AGE. 1985. Histochemistry: theoretical and applied. Vol II. Edinburgh, Livingstone. ); and Coomassie brilliant blue (Fisher 1968Fisher DB. 1968. Protein staining of ribboned epon sections for light microscopy. Histochemistry Cell Biology 16: 92-96.) 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 1965Karnovsky MJ. 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. Journal Cell Biology 27 137-138.) for 48 h. After dehydration in an ethanol series, the specimens were critical point-dried using CO2 (Autosamdri®, 815, Séries A) (Bozzola & Russel 1992Bozzola JJ, Russel LD. 1992. Electron microscopy. Boston, Jones and Bartlett Publishers.), 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).

Results

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).

Figure 2
Colleters in Clusia criuva Cambess. subsp. criuva. A-C. Abundant secretion (asterisk) covering the leaf primordia (lp). D-F. Colleters on young leaves. D. Brownish color at the apex of the colleters. E. Constriction at the apical portion (arrows). F. Cuticle rupture (arrowheads). G. Extravasated secretion (asterisks). H, I. Colleters on adult leaves. H. Accentuated brown color. I. Collapsed cell, reduced secretion (asterisks) and constriction at the apex of the colleter (arrows). J-L. Colleters in senescent leaves. J. Blackened colleters. K. Collapsed colleters. L. Cuticle rupture (arrowheads). Abbreviation: co = colleters. Bars: A, D, H = 2 mm; B, C, I = 500 μm; E, L = 100 μm; F = 20 μm; G = 2 μm; J = 2 mm; K = 200 μm.

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.

Figure 3
Anatomical sections of the leaf colleters of Clusia criuva Cambess. subsp. criuva. A-F. Colleters in early leaf. A. Asynchronous development from protodermal and subepidermal cells. B. Detail of the undifferentiated cells of the colleters, which have an evident nucleus and nucleolus. C. Intense vacuolization (stars). D. Colleter with central axis and secretory epidermis. E. Standard type colleters, sessile with multiseriate epidermis. F. Non-vascularized colleter. G. Cuticle stained with Sudan III. H, I. Colleters starting senescence (arrowheads). J. Senescent colleter. Asterisks indicate secretion. Abbreviations: ca = central axis; se = secretory epidermis. Bars: A, D, I, J = 500 μm; E, F, H = 200 μm; B, C = 20 μm; G = 50 μm.

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).

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.

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 (2006Evert RF. 2006. Esau’s Plant anatomy: meristems, cells, and tissues of the plant body: their structure, function, and development, 3rd. edn. Hoboken, John Wiley & Sons.). In Ilex species, González (1998González AM. 1998. Colleters in Turnera and Piriqueta (Turneraceae). Botanical Journal of the Linnean Society 128: 215-228.) 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 2006Paiva EAS, Machado SR. 2006. Colleters in Caryocar brasiliensis(Caryocaraceae), ontogenesis, ultrastructure and secretion. Brazilian Journal of Biology 66: 301-308.; Paiva 2009Paiva EAS. 2009. Occurrence, structure and functional aspects of the colleters ofCopaifera langsdorffiiDesf. (Fabaceae, Caesalpinioideae). Comptes Rendus Biologies 332: 1078-1084.; Paiva & Martins 2011Paiva EAS, Martins LC. 2011. Calycinal trichomes in Ipomoea cairica (Convolvulaceae): ontogenesis, structure and functional aspects. Australian Journal of Botany 59: 91-98.; Rocha et al. 2011Rocha JF, Pimentel RR, Machado SR. 2011. Estruturas secretoras de mucilagem em Hibiscus pernambucensis Arruda (Malvaceae): distribuição, caracterização morfoanatômica e histoquímica. Acta Botanica Brasilica 25: 751-763.). 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 1981Machado MMP, Emmerich M. 1981. Presença de coléteres em Clusia lanceolata Cambess. Boletim do Museu Nacional de Botânica 59: 1-7.; Silva et al. 2019Silva KMM, Luna BN, Joffily A, Paiva SR, Barros CF. 2019. Revealing the development of secretory structures in the leaves of Clusia fluminensis and Clusia lanceolata (Clusiaceae). Flora 256: 69-78.) and C. burchellii (Teixeira et al. 2021Teixeira RS, Rocha DI, Dalvi V. 2021. Leaf colleters in Clusia burchellii Engl.: Structural and ultrastructural features of a little-known gland in Clusiaceae. Flora 280: 151834.), 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 (1974Lersten NR. 1974. Morphology and distribution of colleters and crystals in relation to the taxonomy and bacterial leaf nodule symbiosis of Psychotria (Rubiaceae). American Journal of Botany 61: 973-981.). The standard type colleter also was found in C. grandiflora (Machado & Emmerich 1981Machado MMP, Emmerich M. 1981. Presença de coléteres em Clusia lanceolata Cambess. Boletim do Museu Nacional de Botânica 59: 1-7.) and C. burchelli (Teixeira et al. 2021Teixeira RS, Rocha DI, Dalvi V. 2021. Leaf colleters in Clusia burchellii Engl.: Structural and ultrastructural features of a little-known gland in Clusiaceae. Flora 280: 151834.). In C. fluminensis and C. lanceolata, lachrymiform cone-shaped colleters were described (Silva et al. 2019Silva KMM, Luna BN, Joffily A, Paiva SR, Barros CF. 2019. Revealing the development of secretory structures in the leaves of Clusia fluminensis and Clusia lanceolata (Clusiaceae). Flora 256: 69-78.). 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)Lersten NR. 1974. Morphology and distribution of colleters and crystals in relation to the taxonomy and bacterial leaf nodule symbiosis of Psychotria (Rubiaceae). American Journal of Botany 61: 973-981.. 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 2006Paiva EAS, Machado SR. 2006. Colleters in Caryocar brasiliensis(Caryocaraceae), ontogenesis, ultrastructure and secretion. Brazilian Journal of Biology 66: 301-308.; Silva et al. 2017Silva MDS, Coutinho ÍAC, Araújo MN, Meira RMSA. 2017. Colleters in Chamaecrista (L.) Moench sect. Chamaecrista and sect. Caliciopsis (Leguminosae-Caesalpinioideae): anatomy and taxonomic implications. Acta Botanica Brasilica 31: 382-391.).

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 1988Fahn A. 1988. Plant anatomy. Oxford, Pergamon Press.) together with pectin may act as a water absorber due to its hygroscopic properties (Hall 1981Hall WC. 1981. Biomass as an alternative fuel. Rockville, Government Institutes Inc.; Nobel et al. 1992Nobel PS, Alm DM, Cavelier J. 1992. Growth respiration, maintenance respiration and structural-carbon costs for roots of three desert succulents. International Journal of Plant Sciences 153: 102-107.), 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 1968Lersten NR, Horner HTJR. 1968. Development, structure and function of secretory trichomes in Psychotria bacteriofila(Rubiaceae). American Journal of Botany 55: 1089-1099.; Lersten 1974Lersten NR. 1974. Morphology and distribution of colleters and crystals in relation to the taxonomy and bacterial leaf nodule symbiosis of Psychotria (Rubiaceae). American Journal of Botany 61: 973-981.; Fahn 1979Fahn A. 1979. Secretory Tissues in Plants. London, Academic Press.) and/or that promote the lubrication of the stem meristem, minimizing friction of the developing leaf tissues and preventing dehydration (Fahn 1979Fahn A. 1979. Secretory Tissues in Plants. London, Academic Press.; Thomas 1991Thomas V. 1991. Structural, functional and phylogenetic aspects of the colleter. Annals of Botany 68: 287-305.; Mayer et al. 2011Mayer JLS, Cardoso-Gustavson P, Appezzato-Da-Glória B. 2011. Colleters in monocots: New record for Orchidaceae. Flora 206: 185-190.; Silva et al. 2012Silva CJ, Barbosa LCDA, Marques AE, Baracat-Pereira MC, Pinheiro AL, Meira RMSA. 2012. Anatomical characterisation of the foliar colleters in Myrtoideae (Myrtaceae). Australian Journal of Botany 60: 707-717.; Mayer et al. 2013Mayer JLS, Carmello-Guerreiro SM, Mazzafera P. 2013. A functional role for the colleters of coffee flowers. AoB Plants 5: 1-13.). Proteins are involved in the defense against fungal activity and attack of pathogens (Klein et al. 2004Klein DE, Gomes VM, Silva-Neto SJ, Cunha M. 2004. The structure of colleters in several species ofSimira(Rubiaceae). Annals of Botany 94: 733-740.; Miguel et al. 2006Miguel EC, Gomes VM, Oliveira MA, Cunha M. 2006. Colleters in Bathysa nicholsonii K. Schum. (Rubiaceae): Ultrastructure, secretion protein composition, and antifungal activity. Plant Biology 8: 715-722.) and are commonly reported in the secretion of colleters (González & Tarragó 2009González AM, Tarragó JR. 2009. Anatomical structure and secretion compounds of colleters in nine Ilex species (Aquifoliaceae) from southern South America. Botanical Journal of the Linnean Society 160: 197-210.; Coelho et al. 2013Coelho VPM, Leite JPV, Fietto LG, Ventrella MC. 2013. Colleters inBathysa cuspidata(Rubiaceae): Development, ultrastructure and chemical composition of the secretion. Flora 208: 579-590.; Dalvi et al. 2014Dalvi VC, Cardinelli LS, Meira RMSA, Azevedo AA. 2014. Foliar colleters in Macrocarpaea obtusifolia (Gentianaceae): anatomy, ontogeny, and secretion. Botany 92: 59-67.; Lacchia et al. 2016Lacchia APS, Tölke EEAD, Demarco D, Carmello-Guerreiro SM. 2016. Presumed domatia are actually extrafloral nectaries on leaves of Anacardium humile(Anacardiaceae). Rodriguesia 67: 19-28.; Pinheiro et al. 2019Pinheiro SKP, Teófilo FBS, Lima AKM, Cordoba BV, Miguel TBAR, Miguel EC. 2019. Ontogenesis and secretion mechanism of colleters in Morinda citrifolia L. (Rubiaceae). South African Journal of Botany 121: 26-33.).

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. 2019Silva KMM, Luna BN, Joffily A, Paiva SR, Barros CF. 2019. Revealing the development of secretory structures in the leaves of Clusia fluminensis and Clusia lanceolata (Clusiaceae). Flora 256: 69-78.). According to Coelho et al. (2013Coelho VPM, Leite JPV, Fietto LG, Ventrella MC. 2013. Colleters inBathysa cuspidata(Rubiaceae): Development, ultrastructure and chemical composition of the secretion. Flora 208: 579-590.), 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. 2019Pinheiro SKP, Teófilo FBS, Lima AKM, Cordoba BV, Miguel TBAR, Miguel EC. 2019. Ontogenesis and secretion mechanism of colleters in Morinda citrifolia L. (Rubiaceae). South African Journal of Botany 121: 26-33.), 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. 2021Teixeira RS, Rocha DI, Dalvi V. 2021. Leaf colleters in Clusia burchellii Engl.: Structural and ultrastructural features of a little-known gland in Clusiaceae. Flora 280: 151834.) and species from other families, including Apocynaceae (Thomas & Dave 1989Thomas V, Dave Y. 1989. Structure, origin, development and senescence of colleters in Nerium indicum mill. (Nerium odorum Soland, Apocynaceae). Korean Journal Botany 32: 163-172.; Appezzato-da-Glória & Estelita 2000Appezzato-Da-Glória B, Estelita MEM. 2000. Development, structure and distribution of colleters in Mandevilla illustris and M. velutina (Apocynaceae). Revista Brasileira de Botânica 23: 113-120. ; Rio et al. 2002Rio MCS, Castro MM, Kinoshita LS. 2002. Distribuição e caracterização anatômica dos coléteres foliares dePrestonia coalita(Vell.) Woodson (Apocynaceae). Revista Brasileira de Botânica 25: 339-349.), Gentianaceae (Dalvi et al. 2013Dalvi VC, Meira RMSA, Francino DMT, Silva LC, Azevedo AA. 2013. Anatomical characteristics as taxonomic tools for the species of Curtia and Hockinia (Saccifolieae-Gentianaceae Juss.). Plant Systematics and Evolution 300: 99-112.; 2014Dalvi VC, Cardinelli LS, Meira RMSA, Azevedo AA. 2014. Foliar colleters in Macrocarpaea obtusifolia (Gentianaceae): anatomy, ontogeny, and secretion. Botany 92: 59-67.), and Rubiaceae (Lersten 1974Lersten NR. 1974. Morphology and distribution of colleters and crystals in relation to the taxonomy and bacterial leaf nodule symbiosis of Psychotria (Rubiaceae). American Journal of Botany 61: 973-981.; Miguel et al. 2006Miguel EC, Gomes VM, Oliveira MA, Cunha M. 2006. Colleters in Bathysa nicholsonii K. Schum. (Rubiaceae): Ultrastructure, secretion protein composition, and antifungal activity. Plant Biology 8: 715-722.; 2009Miguel EC, Moraes DG, Cunha M. 2009. Stipular colleters in Psychotria nuda (Cham. & Schltdl.) Wawra (Rubiaceae): micromorphology, anatomy and cristals microanalysis. Acta Botanica Brasilica 23: 1034-1039.; Vitarelli & Santos 2009Vitarelli NC, Santos M. 2009. Anatomia de estípulas e coléteres de Psychotria carthagenensis Jacq. (Rubiaceae). Acta Botanica Brasilica 23: 923-928.; Klein et al. 2010Klein DE, Oliveira MA, Cunha M. 2010. Ultrastructure of secretory and senescence phase in colleters of Bathysa gymnocarpa and B. stipulata (Rubiaceae). Revista Brasileira de Botânica 33: 425-436.; Pinheiro et al. 2019Pinheiro SKP, Teófilo FBS, Lima AKM, Cordoba BV, Miguel TBAR, Miguel EC. 2019. Ontogenesis and secretion mechanism of colleters in Morinda citrifolia L. (Rubiaceae). South African Journal of Botany 121: 26-33.). 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. 2017Ribeiro JC, Ferreira MJP, Demarco D. 2017. Colleters in Asclepiadoideae (Apocynaceae): protection of meristems against desiccation and new functions assigned. International Journal of Plant Sciences 178: 465-477.), which would act to avoid predation (Castro & Demarco 2008Castro MM, Demarco D. 2008. Phenolic compounds produced by secretory structures in plants: a brief review. Natural Product Communications 3: 1273-1284.). 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.

Acknowledgments

We thank the Ministério da Ciência e Tecnologia/Conselho Nacional de Desenvolvimento Científico e Tecnológico (Grant #406824/2016-9) and the IF Goiano, campus Rio Verde, for financial support; the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Finance Code 001, for the Master's scholarship granted to first author; the Laboratório Multiusuário de Microscopia de Alta Resolução (LabMic/UFG) for the preparation and analysis of electron microscopy samples; Fernando Henrique Antoniolli Farache for creating the map, and Patrícia Oliveira da Silva for the drawings in Figure 1.

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Publication Dates

  • Publication in this collection
    24 June 2022
  • Date of issue
    2022

History

  • Received
    23 Mar 2021
  • Accepted
    05 Feb 2022
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