Botany

Structure and function of secretory glochids and nectar composition in two (Cactaceae) species

Journal: Botany

Manuscript ID cjb-2020-0004.R2

Manuscript Type: Article

Date Submitted by the 06-Apr-2020 Author:

Complete List of Authors: Silva, Stefany; Universidade Estadual Paulista Julio de Mesquita Filho, Botany Machado, Silvia; UNESP Nepi, Massimo; Università degli Studi di Siena Rodrigues,Draft Tatiane; UNESP, Botany Keyword: anatomy, , nectar, secretory spines, ultrastructure

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Structure and function of secretory glochids and nectar composition in two

Opuntioideae (Cactaceae) species

Stefany Cristina de Melo Silva, Silvia Rodrigues Machado, Massimo Nepi, Tatiane Maria

Rodrigues

S.C.M. Silva

UNESP - São Paulo State University, Institute of Biosciences, Department of Botany, 18618-

970, Botucatu city, São Paulo State, Brazil

S.R. Machado UNESP - São Paulo State University,Draft Institute of Biosciences, Department of Botany, 18618- 970, Botucatu city, São Paulo State, Brazil

Massimo Nepi

University of Siena, Department of Life Sciences, 53100, Siena, Italy.

T.M. Rodrigues

UNESP - São Paulo State University, Institute of Biosciences, Department of Botany, 18618-

970, Botucatu city, São Paulo State, Brazil

Corresponding author: Tatiane Maria Rodrigues (e-mail: [email protected])

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ABSTRACT

Cactaceae exhibit highly modified spines considered as extrafloral nectaries (EFNs). Despite their ecological and taxonomical relevance in this family, little is known on their structure and function. We described the anatomy, ontogenesis, and ultrastructure of the secretory glochids in two Opuntioideae species. Young cladodes of Brasiliopuntia brasiliensis and Nopalea cochenillifera were processed for light and electron microscopy studies. The secretion composition was analyzed by high performance liquid chromatography. Secretory glochids were soft, massive and barbed, as well as translucent. Hyaline droplets on the secretory glochid apex were collected by aggressive ants. Secretory glochids originated from the areolar meristem, beginning as small protuberances formed by protoderm and ground meristem.

Mature secretory glochids consisted of a central multiseriate axis of ground cells covered by uniseriate epidermis with continuousDraft cuticle, and exhibited three regions: i) dilated vascularized base with parenchyma cells exhibiting features associated to nectar secretion; ii) elongated median region with juxtaposed fusiform non-lignified parenchyma cells; and iii) tapered apical portion with immature fibers loosely arranged cells. The exudate was sucrose- dominant with a similar amino acid profile in both species. Our results shed light on the secretory activity of glochids in Cactaceae and their role in cactus-ant interactions.

Keywords: anatomy; Brasiliopuntia brasiliensis; cactus; nectar; Nopalea cochenillifera; secretory spines; ultrastructure

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Introduction

Cladodes, succulent stems bearing areoles with spines, are a synapomorphy of

Cactaceae (Nyffeler 2002; Judd et al. 2008). Two different morphological categories of spines

are described in this family: larger and rigid spines consisting exclusively of lignified cells

along their entire length; and smaller, hyaline and flexible spines covered by epidermis with

beard-shaped cells and containing a non-lignified basal region, called glochids. The first type

is widespread in Cactaceae, while glochids are restricted to Opuntioideae members (Gibson

and Nobel 1986; Mauseth 2006).

In several members of Cactaceae, including Opuntioideae, highly modified spines are

involved in nectar secretion and have been considered as extrafloral nectaries (EFNs) (Díaz-

Castelazo et al. 2005; Mauseth et al. 2016; Sandoval-Molina et al. 2018). However, detailed studies on the EFN structure in CactaceaeDraft are available for only few taxa and even species with the most obvious and highly modified glands are poorly studied (Mauseth et al. 2016).

The morphology and localization of EFNs in Cactaceae have systematic value (Mauseth et al.

2016; Sandoval-Molina et al. 2018). In addition, the ecological importance of EFNs in

members of the family has been emphasized in studies showing mutualistics interactions with

ants and the secretion seasonality (Pickett and Clark 1979; Blom and Clark 1980; Oliveira et

al. 1999; Ness 2006). Studies on the morphogenesis, structure and functioning of the EFNs in

Cactaceae can present high added value, providing support and information for

(Mauseth et al. 2016) and ecological studies (Díaz-Castelazo et al. 2005; Sandoval-Molina et

al. 2018).

The structure of EFNs and nectar composition affects the attractiveness of to

nectar-foraging insect visitors (Diaz-Castelazo et al. 2005, 2017). In general, the

morphological and anatomical complexity of the EFNs is linked to their vascularization

degree, being that the proportion of phloem and xylem in the EFNs reflects in the nectar

concentration, i.e., the more developed the phloem, the more concentrated the nectar is (Fahn

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1979; Nepi 2007). Vascularized EFNs in Opuntioideae were reported only in stricta

(Oliveira et al. 1999), while non-vascularized EFNs seem to be predominant among this subfamily (see Sandoval-Molina et al. 2018 and cited references). The nectar from EFNs in

Opuntioideae species is composed of sugars such as glucose, fructose and sucrose commonly found in the exudate of many other species; however, the amino acid concentration seems to be higher than what has been reported in the nectar from other plant groups (Pickett and Clark 1979).

In order to understand the function of the secretory glochids, we conducted histological, cytological and chemical analyses in Brasiliopuntia brasiliensis (Willd.) A.

Berger and Nopalea cochenillifera (L.) Salm-Dyck, two members of Opuntioideae co- occurring in the Atlantic Forest. Draft Materials and Methods

Plant species

Brasiliopuntia brasiliensis and Nopalea cochenillifera are shrubby species occurring in the Caatinga (an ecological region of northeastern Brazil occupied by tropical dry forest and scrub vegetation) and Atlantic Forest (Zappi and Taylor, in prep). We sampled adult individuals of B. brasiliensis (n = 4) and N. cochenillifera (n = 5) cultivated in the experimental area of the Department of Botany of the Institute of Biosciences of Botucatu

(IBB), São Paulo State University (UNESP), Botucatu city (22°53’08” S, 48°26’42” W), São

Paulo State, Brazil.

The ants observed collecting nectar from EFNs were collected, stored in 70% alcohol and identified by Dr. Roberto da Silva Camargo of the Laboratory of Social Insects and Pests,

Faculty of Agronomic Sciences (FCA), UNESP, Botucatu.

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Vouchers were deposited in the Herbarium Irina Delanova Gemtchúnicov (BOTU),

Department of Botany, IBB, UNESP, under the register numbers 33069 and 33070 for B.

brasiliensis and 32966, 32967 and 33071 for N. cochenillifera. The plant identification was

confirmed by Dr. Lidyanne Yuriko Saleme Aona of the Universidade Federal do Recôncavo

da Bahia.

Light Microscopy

EFNs in the secretory stage (swollen and containing exudate drops on their apex) were

observed using a Leica M205C stereomicroscope. Images were recorded using a Leica DFC

425 digital camera coupled to the stereomicroscope. The length of the secretory spines was

measured using a digital caliper. For histological and ontogeneticDraft studies, EFNs from cladodes at different developmental stages (from 3 to 10 cm of length) were collected, fixed in FAA 50

(formaldehyde 37%, acetic acid glacial and ethanol 50%) (Johansen 1940), dehydrated in an

ethanol series and embedded in Leica methacrylate resin. Transverse and longitudinal sections

(5μm thickness) were obtained serially using a Leica RM2245 semiautomatic rotating

microtome and stained with 0.05% Toluidine Blue pH 4.7 (O’Brien et al. 1964). Permanent

slides were mounted using synthetic resin Permount and analyzed using a Leica DMR light

microscope under bright field and under polarized light. The results were documented using a

Leica DFC425 digital camera.

For identification of the main classes of compounds present in the cells of the EFNs,

areolar samples (n = 4) of fresh material were sectioned by hand using a razor blade. The

sections were treated with Fehling reagent for the identification of reducing sugars (Sass

1951); Ruthenium red 0.02% for acid polysaccharides and mucilage (Jensen 1962); periodic

acid / Schiff reagent (PAS) for polysaccharides (Taboga and Vilamaior 2001); Lugol reagent

for starch grains (Johansen 1940); Sudan IV for total lipids (Johansen 1940); bromophenol

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mercuric blue for proteins (Mazia et al. 1953); ferric chloride 10% for phenolic compounds

(Johansen 1940) and hydrochloric acid 10% for calcium oxalate crystals (Chamberlain 1932).

The control tests were performed according to the descriptor of each technique. The material was analyzed using a Leica DMR light microscope and documented with a Leica DFC425 digital camera. For detection of acidic polysaccharides, samples treated with Acridin orange

(Armstrong 1956) were analyzed using a Leica SP5 laser scanning confocal microscope, set at

460 nm excitation and 650 nm emission.

Electron Microscopy

Scanning Electron Microscopy (SEM) - Areolar samples (n = 3) bearing EFNs were fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer pH 7.3, postfixed in 1% osmium tetroxide, dehydrated in a graduate Draftacetone series, dried at a critical point and metallized with gold (Robards 1978). The samples were analyzed using a scanning electron microscope

(SEM), EVO LS15 ZEISS microscopy at 10 kV.

Transmission Electron Microscopy (TEM) – Samples (n = 3) of EFNs in the secretory stage were fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer, pH 7.3, post-fixed in 1% osmium tetroxide in the same buffer, dehydrated in an acetone series and embedded in

Araldite (Machado and Rodrigues 2004). Semi-thin sections were obtained from the basal, median and apical regions of the secretory glochids. The ultrathin sections (70 nm) were contrasted with uranyl acetate (Watson 1958) and lead citrate (Reynolds 1963). For endomembrane system impregnation, samples were processed using the ZIO (Zinc Iodide-

Osmium Tetroxide) technique (Reinecke and Walther 1978). After fixation in 2.5% glutaraldehyde, the samples were incubated in a solution containing Zn, I, TRIS- aminomethane and 1.0% osmium tetroxide. Afterwards, the samples were processed according to the conventional technique for TEM analysis. For detection of carbohydrates, the material was processed according to Thiery (1967). Ultrathin sections were treated with a

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1% thiosemicarbazide solution for 72 hours. Afterwards, the sections were submitted to a

decreasing concentration of acetic acid and incubated with 1% silver proteinate for 30

minutes. The samples were analyzed using a Fei Tecnai Spirit transmission electron

microscopy at 80 kV.

Sampling and Chemical Analysis of the Exudate

Exudate from the EFNs (n = 5) of each species was collected using paper wicks

(WhatmanTM filter paper grade 1, Sigma-Aldrich) (Amato and Petit 2017). Wicks were

weighed before and after exudate collection. After drying at room temperature, the paper

fragments were stored at -20ºC. Samples were thawed in an oven at 40ºC and the exudate was

removed from the filter paper fragments by static elution in distilled water, followed by centrifugation for 5 minutes at Draft 11000g. In order to determine the sugar content, the supernatant was analyzed by isocratic high performance liquid chromatography (HPLC) using

an LC1 system (Waters). Aliquots of 20µL of the sample and the standard solution were

injected. Ultra-pure water (MilliQ, pH 7) in flow of 0.5ml for 21 minutes was used as the

mobile phase. The sugars were separated in a Waters-Sugar Pack I column (6.5-300mm),

maintained at 90ºC and identified with a refractive index detector (Waters 2410) (Stahl et al.

2012).

Amino acid analysis was performed by high HPLC using a Novapak C18 ion exchange

column (15mm x 4.6mm) maintained at 37°C and a Waters 470 fluorescence detector

(excitation at 295nn, detection at 350nm) (Nepi et al. 2012). Solvent composed of TEA-

phosphate buffer pH 5.0 and acetonitrile solution in water (6: 4) was used as the mobile phase

in a flow of 1.0 ml per minute. The aliquots (10µl) of each reconstituted samples were

derivatized with fluorescent reagent AQC (6-aminoquinolyl-N-hydroxysuccinimidyl

carbamate) and 0.02M borate buffer, pH 8.6.

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Results

Areola morphology and field observations

Reduced leaves, non-glandular hairs, long lignified spines, and secretory and non-secretory glochids (Fig. 1A-C) occurred blended in each of the areoles of the young vegetative stems of

B. brasiliensis and N. cochenillifera. In both species, secretory glochids were soft, massive and barbed, with translucent aspect and numbered one to two per areole (Fig. 1B, 1C); they were bigger (c.a. 2mm in length) than the three to six non-secretory glochids (0.6 ± 0.2 mm in length) present in each areole. Secretory and non-secretory glochids were deciduous and abscission of secretory glochids occurred earlier than non-secretory ones in both species.

In B. brasiliensis and N. cochenillifera, hyaline drops were observed on the apex of the secretory glochids (Fig. 1B, 1C).Draft Individual secretory glochids were active for 2-3 consecutive days. The exudate released from the secretory glochids was collected by aggressive ants. Crematogaster sp. and Cephalotes sp. ants were observed moving on the cladodes of both species; Camponotus sp. ants were observed exclusively in B. brasiliensis and Pheidole sp. only in N. cochenillifera. Ants were able to collect the exudate from the apex of the secretory glochids using their mouth parts (Fig. 1D).

Ontogenesis and histology of the secretory glochids

In both species, the secretory glochids originated from the areolar meristem (Fig. 2A).

Secretory glochids were initiated as small emergences covered by protodermis and filled with

2-4 layers of ground meristem cells with isodiametric shape, dense cytoplasm and large nucleus (Fig. 2A). These cells proliferated and produced a large dome-shaped secretory glochid primordium (Fig. 2B). Procambial traces from the cladodium reached the base of the glochid primordium (Fig. 2B). The developing secretory glochids became conical in shape and exhibited a sharper apex and wider base (Fig. 2C). At the base of the secretory glochids,

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the cells were juxtaposed and isodiametric with a large nucleus and abundant dense

cytoplasm; in the apex, the cells became slightly axially elongated.

As the differentiation progressed, three distinct regions (basal, median and apical)

were distinguished (Fig. 2D). In the basal region, the epidermal and ground parenchyma cells

were isodiametric, with dense cytoplasm, large nucleus and moderately developed vacuome

(Fig. 2E). In the median region, the cells retained the dense cytoplasm; the epidermal cells

maintained their isodiametric shape, and the parenchyma cells became fusiform (Fig. 2F). In

the apical portion, the ground cells were immature fibers; epidermal and ground cells

exhibited non-lignified thick walls, reduced cytoplasm and developed vacuome (Fig. 2G); in

this region, the epidermal cells exhibited an imbricate disposition and their lower pole became

detached resembling barbs (Fig. 2D, 2G). Mature glochids active in secretionDraft exhibited fusiform-shaped epidermal cells with tapered edges coated with a smooth cuticle (Fig. 3A, 3B). Cuticular pores or openings were

not observed (Fig. 3B). In apical (Fig. 3C) and median (Fig. 3D) regions of the secretory

glochids, epidermal and ground cells maintained characteristics similar to the previous stage.

In the apical region, the lower pole of each epidermal cell underwent a slight detachment and

protruded (Fig. 3C). Xylem and phloem elements were restricted to the basal region of the

secretory glochids (Fig. 3A, 3E, 3F). Druses of calcium oxalate occurred in the basal region of

the secretory glochids (Fig. 3E). At this stage of development, the secretory glochids tested

positively for detection of reducing sugars, neutral and acid polysaccharides and proteins

(Table 1). Starch grains were detected in the parenchyma cells subjacent to the glochids,

especially near the vascular tissues.

In the post-secretory stage, the cells of the median and apical portions of the glochids

were elongated and had thicked lignified walls (Fig. 4A, 4B). Subjacent to the secretory

glochid basis, several layers of tangentially thin-walled elongated cells with dense cytoplasm,

constituting a basal meristematic zone, were observed (Fig. 4C).

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Ultrastructure and cytochemistry of the secretory glochids

In both species, basal and median regions of the glochids active in secretion were covered by epidermal cells with protruded enlarged ends that overlapped the neighboring cells

(Fig. 5A, 5B). Epidermal cells exhibited thin walls with irregular electron-density, large nucleus, dense cytoplasm and poorly developed vacuome (Fig. 5A, 5B). Large mitochondria, rough endoplasmic reticulum, polysomes, dictyosomes and plastids with poorly developed thylakoids containing dark globules were observed in the cytoplasm (Fig. 5A-D) of the epidermal cells. Vesicles occurred adjacent to the plasmalemma (Fig. 5D). In the apical region of the secretory glochids, the epidermal cells exhibited protruding detached barbed portions (Fig. 5E) as observed in SEM (Fig. 3B). The outer periclinal cell wall consisted of cellulose microfibrils in a loose arrangementDraft forming fenestrae (Fig. 5F). Electron-dense ramifications from the cell wall matrix protruded toward the cuticle (Fig. 5F, G). In the apical region of secretory glochids, the epidermal cells had reduced cytoplasm and a large central vacuole (Fig. 5E).

In the basal region of the secretory glochids the parenchyma cells were compactly arranged and exhibited an irregular contour with portions that protruded toward the neighboring cells (Fig. 6A), their walls were sinuous, and irregular in thickness and electron density (Fig. 6A-C). In the cell corners, the middle lamella was swollen (Fig. 6A, 6B).

Plasmodesmata were abundant and connected the parenchyma cells (Fig. 6D). Parenchyma cells exhibited dense cytoplasm, large nucleus and small vacuoles filled with flocculent material (Fig. 6A-C). Plastids containing globular dark inclusions (Fig. 6B) or oval starch grains (Fig. 6C) and dictyosomes with fenestrae (Fig. 6D, 6E) were common in these cells.

ZIO reaction products appeared as dense deposits in the lumen of the outer cisternae of the

Golgi bodies on their cis and trans faces, and in many vesicles attached to their ends or near them (Fig. 6D, 6E). Positive reaction for polysaccharides (seen as fine dense granules) was

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observed in the cell walls, cytosol, vacuoles (Fig. 6F) and inside the plastids (Fig. 6G).

Strands of phloem containing sieve tube members and companion cells, and parenchyma cells

were observed in the basal region of the secretory glochids (Fig. 6H).

In the median region of the secretory glochids, the fusiform parenchyma cells were

loosely arranged giving rise to intercellular spaces (Fig. 7A). Cell walls bearing

plasmodesmata, large nucleus, dense cytoplasm rich in mitochondria and plastids with poorly

developed thylakoids, several vacuoles filled with flocculent material (Fig. 7A-C) and

scattered actin strands (Fig. 7D) in the cytosol characterized the morphology of the

parenchyma cells.

In the apical region, the secretory glochids were filled with immature fibers with

centrifugal differentiation. The cells subjacent to the epidermis exhibited an irregular contour, thicker walls, reduced cytoplasm,Draft and well-developed vacuoles. Prominent spaces were observed among them (Fig. 7E). In the central region of the glochids, small spaces occurred

among the cells that were hexagonal in cross section (Fig. 7F) and fibriform in longitudinal

section (Fig. 7G). Plasmodesmata were common in their thick walls (Fig. 7H). In the central

region of the glochid, the cells with greatly reduced or even absent cytoplasm occurred

interspersed with cells exhibiting small vacuoles (Fig. 7I) and dense cytoplasm (Fig. 7F, G)

with mitochondria, rough endoplasmic reticulum, plastids with lipid inclusions.

Chemical analysis of secretion

Glucose, fructose and sucrose were the carbohydrates detected in the exudate of the

secretory glochids (Table 2). In both species, the exudate was sucrose-dominant. The total

sugar concentration was higher in N. cochenillifera than in B. brasiliensis. The proportion of

sucrose in the exudate was similar between the studied species (Table 2).

Several amino acids were identified in the exudate released from the secretory

glochids in B. brasiliensis and N. cochenillifera; the highest total concentration was observed

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in B. brasiliensis (Table 3). In general, the composition of amino acids was similar in both species; however, leucine was detected only in N. cochenillifera and lysine only in B. brasiliensis exudates. Histidine was the most abundant and glycine the least abundant amino acid in the exudate of both species (Table 3).

Discussion

The secretory glochids of Brasiliopuntia brasiliensis and Nopalea cochenillifera are highly modified spines with an enlarged secretory base, and fits into one of the four EFNs morphotypes described by Mauseth et al. (2016).

The initiation and development of the secretory glochids in B. brasiliensis and N. cochenillifera were very similar. In both the species, the secretory glochids arosen from the areolar meristem and exhibited basipetalDraft and centrifugal differentiation, as observed in lignified spines and non-secretory glochids of other Cactaceae species (Boke 1944, 1951;

Gibson and Nobel 1986; Arruda and Melo-de-Pinna 2016). Glochids of B. brasiliensis and N. cochenillifera active in secretion consisted of cells with non-lignified walls and dense protoplast. In their post-secretory stage, glochids became completely lignified, except at their basal region as reported for non-secretory glochids of other Cactaceae species (Gibson and

Nobel 1986; Arruda and Melo-de-Pinna 2016). However, even after the end of their secretory activity, the secretory glochids of B. brasiliensis and N. cochenillifera may continue to elongate due to the permanence of an intercalary meristem subjacent to their base.

The mature secretory glochids of B. brasiliensis and N. cochenillifera exhibited three morphologically distinct regions: basal, median and apical and our histological and cytological analyses enabled us to suggest that each one can act differently in the secretory process. In this sense, the basal region of the secretory glochids in both the species is responsible for nectar production, while the median is involved in the transport of the exudate toward the apical portion of the glochids, where the secretion is released to the outside

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through the intact cuticle. In fact, the cellular features exhibited by the basal region, such as

small size, thin walls, large nucleus and dense cytoplasm rich in mitochondria, dictyosomes,

plastids and endoplasmic reticulum, is typical of nectar-secreting structure (Evert 2006; Nepi

2007). In these cells, the ZIO method enabled us to identify the dictyosomal origin of the

vesicles in the cytoplasm, while the Thiéry method revealed the accumulation of

polysaccharides in the cell walls and in the cytoplasm of the secretory cells. In addition, the

presence of reducing sugars (identified using Fehling reagent) and polysaccharides (identified

using Acridine orange and PAS) were histochemically detected in these cells. The

ultrastructural features of the parenchyma cells in combination with the results of the

histochemical tests confirm the involvement of the basal region of the glochids in nectar

production and indicate that this region corresponds to the “specialized parenchyma tissue” (Fahn 1979) or “nectariferous parenchyma”Draft (Nepi 2007) described in the literature for other conventional types of nectaries.

In B. brasiliensis and N. cochenillifera, the pre-nectar in the secretory glochids can

come from two distinct sources, i.e., starch grains and phloem sap. The occurrence of starch

grains in the cladode parenchyma cells subjacent to the basal region of the secretory glochids

indicates that these cells are the source of carbohydrates for the production of nectar.

However, the abundance of starch grains in these parenchyma cells is lower when compared

to the subnectariferous parenchyma of some species with other types of nectaries (Fahn 1979;

Paiva and Machado 2008). Following Pacini et al. (2003), the carbohydrates participating in

the nectar composition are not always stored as starch grains; these carbohydrates can be

immediate derivatives of the photosynthesis carried out in any part of the plant. In fact, the

presence of phloem and xylem in the basal region of the secretory glochids in B. brasiliensis

and N. cochenillifera suggest that the pre- nectar can be transported by the vascular tissues

from other regions of the cladode.

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Among the Opuntioideae, the presence of vascular tissues in the secretory spines is not a unifying character. Vascularized EFNs are unknown in species from Opuntioideae subfamily (Sandoval-Molina et al. 2018), having been reported only in O. stricta (see Diaz-

Castelazo et al. 2005 and literature therein). Such scarcity of reports of vascularized EFNs in

Opuntioideae can be attributed to the low number of studied species and to methodological difficulties in accessing the vascular tissues in their massive basal region. In fact, a large amount of samples was needed to be processed and analyzed in order to reach the vascular tissues in the secretory glochids of N. cochenillifera and B. brasiliensis. Vascular tissues are particularly important in nectaries by facilitating the arrival of substances that will be used in the production of nectar (Evert 2006) and the concentration of nectar produced is associated with the vascularization that the nectary receives (see Nepi 2007 and cited literature). The presence of cells with sinuousDraft pecto-cellulosic walls and intrusive growth, and the swollen middle lamellae in the corner of the cells in the basal region of the secretory glochids in B. brasiliensis and N. cochenillifera probably favors the movement of hydrophilic substances through the cell wall. These features are consistent with apoplastic pathways of nectar transport. In addition, the projection of cell ends toward neighboring cells in a model- like intrusive growth (Machado et al. 2017) may facilitate transport of secretions between neighboring cells. The abundance of plasmodesmata in the walls of these cells indicates the occurrence of symplastic transport and is a characteristic described for EFNs of other plant species, including Cactaceae (Sandoval-Molina et al. 2018). The simultaneous apoplastic and symplastic transport pathways of nectar have been observed in nectaries of several species

(Davis et al. 1988; Stpiczynska 2003; Wist and Davis 2006). Our results indicate that the nectar produced in the basal portion of the spines in B. brasiliensis and N. cochenillifera is transported via the symplast and apoplast toward the apical portion of the secretory glochids, from where it is released to the nectary surface through the intact cuticle. In general, the ways by which nectar crosses the cuticle and reaches the outside is still controversial (Paiva 2017).

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In B. brasiliensis and N. cochenillifera, the features of epidermal cells in the apical region

include a loose appearance of microfibrils and electron-dense parietal ramifications that

project towards the cuticle; these features contribute to the increased porosity of the cell wall

to macromolecules (Taiz and Zeiger 1998). Such parietal ramifications consist of

polysaccharidic material from the cell wall protruding into the cuticular layers and represent

hydrophilic pathways (as per Paiva 2017) for the passage of hydrophilic secretions through

the hydrophobic cuticle (Rocha and Machado 2009; Paiva 2017). Considering that modified

stomata and pores or openings are absent on the surface of the secretory glochids of B.

brasiliensis and N. cochenillifera, such hydrophilic pathways seem to be the only route for

nectar release toward the surface of the EFNs, suggesting the permeability of the cuticle to the

secretion (Stpiczynska et al. 2005; Rocha and Machado 2009). In contrast to results reported for Opuntia robusta by Sandoval-MolinaDraft et al. (2018), in B. brasiliensis and N. cochenillifera, the nectar is released through the intact epidermis. Therefore, manipulations of the secretory

glochids by ants do not seem necessary in this process.

The presence of actin strands in the cytoplasm of the cells of the median region of the

secretory spines may be associated with vesicle movements in the cytoplasm of cells

(Valderrama et al. 2001; Stamnes 2002; Tozin and Rodrigues 2017). A positive reaction using

bromophenol blue in this region may be associated with the presence of such protein

filaments in these cells. Following Tozin and Rodrigues (2017), secretory cells in plants show

differential distribution of actin microfilaments and microtubules according to the

composition of secretions produced; according to the authors, actin filaments are more

strongly labeled in cells secreting hydrophilic substances.

The presence of crystals of calcium oxalate in the cladode parenchyma adjacent to the

basal region of the secretory glochids, in both the studied species, seems to be a feature

common to EFNs (Horner et al. 2003; Stpiczynska et al. 2003; Ávila‑Argáez et al. 2018).

Considering that the mechanism of transport of sucrose in plants involves ATPases and that

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excess calcium is able to inhibit the activity of this enzyme, the immobilization of calcium ions in the form of crystals is important for glandular functioning (Nepi 2007). In this sense, the presence of calcium crystals can be indicative of cells active in symplastic transport (Paiva et al. 2007), suggesting that the parenchyma subjacent to the EFN is involved in transferring carbohydrates from the phloem toward the secretory tissue (Wergin et al. 1975; Paiva et al.

2007). In addition, the presence of calcium oxalate crystals may be effective in protecting plant tissues against herbivory (Prychid and Rudall 1999) and can be important in maintaining the integrity of the nectaries, preventing herbivores from feeding on nectary tissues (Gish et al. 2016).

Ants visiting the secretory glochids and removing the exudate, as recorded in both species, is evidence of ant patrols and their interaction with EFNs in Cactaceae (Pickett and Clark 1979; Bloom and Clark 1980;Draft Oliveira et al. 1999; Díaz-Castelazo et al. 2005; Mauseth et al. 2016). The exudate produced by the secretory glochids of B. brasiliensis and N. cochenillifera consisted of an aqueous solution rich in sugars and amino acids, and was also observed in EFNs of other representatives of Cactaceae (Pickett and Clark 1979; Ruffner and

Clark 1986). This nectar composition makes it quite attractive for ants (Lanza 1998; Blüthgen and Fiedler 2004; Byk and Del Claro 2011). In the exudate produced by both species, nine of the nineteen amino acids identified are considered essential amino acids for insect nutrition

(Bentley 1977; Pickett and Clark 1979). The positive labeling for proteins along the body of the glandular glochids may be related to the presence of amino acids in the secretion.

Although the sugar and amino acid profile is very similar in the extrafloral nectar of the two species, they differ considerably in the ratio of sugars to amino acid concentrations. The large amount of carbon (sugars), as detected in the exudate of N. cochenillifera, may increase ants’ desire for N-rich protein and hence the likelihood that they will attack herbivorous insects on the host plant, potentiating their indirect defence (Ness et al. 2009). Here, we suggest that the distinctive trait of nectar chemistry can be related to the differences in the species visiting the

16 https://mc06.manuscriptcentral.com/botany-pubs Page 17 of 39 Botany

EFNs of these two cacti species. Several benefits have been attributed to the association of

ants with extrafloral nectaries, such as shelter and / or food source, which may be associated

with providing plant protection against herbivory, dispersal of seeds and even pollination of

their flowers (Fernández 2003; Rico-Gray and Oliveira 2007). In addition, the ants attracted

by the secretion of EFNs can protect them from harmful organisms such as other insects and

fungi (Ness 2006; Chamberlain and Holland 2008; Mauseth et al. 2016).

Further studies are required to test the extent of the benefits from the mutualistic

relationships to B. brasiliensis and N. cochenillifera and the ant species, and whether these

relations are more widespread than previously thought throughout Cactaceae.

Acknowledgments The authors thank the FundaçãoDraft de Amparo à Pesquisa do Estado de São Paulo - FAPESP - for the scholarship to S.C.M. Silva (Process number 2017 / 14891-5); the Conselho

Nacional de Desenvolvimento Científico e Tecnológico - CNPq - for the research

productivity fellowship granted to T. M. Rodrigues (Process number 303981/2018-0) and

S.R. Machado (Process number 304396/2015-0); Dr. L.S. Aona from Universidade Federal do

Recôncavo da Bahia for the identification of plant species; and Dr. R.S. Camargo from São

Paulo State University for the ant identification. This study was financed in part by the

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil – CAPES (Finance

Code 001).

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1 Figure legends

2

3 Fig. 1 Areoles of young cladodes in Brasiliopuntia brasiliensis (A, B) and Nopalea

4 cochenillifera (C, D). (A) Areole with reduced leaf, lignified spine, secretory glochids

5 and non-glandular hairs. Observe ants visiting the areole. (B) Secretory glochid with

6 liquid exudate droplet at the apex, non- secretory glochids and non-glandular hairs. (C)

7 Secretory glochid with exudate droplet in the apex. (D) Ant removing exudate from the

8 apex of secretory glochid. GG = secretory glochid; HR = non-glandular hairs; LF =

9 reduced leaf; LS = lignified spine; NG = non- secretory glochid. Scale bars: 1 mm (A,

10 D); 0.2 mm (B); 0.5 mm (C).

11

12 Fig. 2 Photomicrographs of the ontogenesisDraft of secretory glochids in Brasiliopuntia

13 brasiliensis. (A) Initial protuberance consisting of protodermal and ground meristem

14 cells. (B) Dome-shaped primordium of secretory glochid consisting of isodiametric

15 cells. (C) Developing secretory glochid with conical shape consisting of cells with

16 dense cytoplasm. (D-G) Developing secretory glochid with distinct basal, median and

17 apical regions. (E) Basal region with isodiametric cells with dense cytoplasm and large

18 nuclei. (F) Median region with isodiametric epidermal cells and slightly elongated

19 parenchyma cells with dense cytoplasm and large nuclei. (G) Apical region with

20 fusiform cells. Note the detached portion of the lower pole of each epidermal cell. AP =

21 apical region; BS = basal region; MD = median region; PC: procambial strand. Scale

22 bars: 50 µm (A, F, G); 100 µm (B, C, E); 150 µm (D).

23

24 Fig. 3 Photomicrographs (A, C-F) and scanning electron micrography (B) of mature

25 secretory glochids of Nopalea cochenillifera (A-C, F) and Brasiliopuntia brasiliensis

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26 (D, E). (A) Secretory glochid with basal, median and apical regions. (B) Glochid

27 surface with fusiform-shaped epidermal cells covered by a smooth cuticle. (C) Apical

28 region with fusiform cells. Observe low density of the cytoplasm in the ground cells and

29 the dense and abundant cytoplasm in the epidermal cells. The lower end of the

30 epidermal cells is detached. (D) Median region with isodiametric epidermal cells with

31 dense cytoplasm and fusiform ground cells with less dense cytoplasm. (E-F) Vascular

32 elements in the basal region of the glochids. Observe the cytoplasmically dense

33 parenchyma cells with developed nucleus with evident nucleolus. DR: druse of calcium

34 oxalate. AP = apical region; BS = basal region; MD = median region; PH: phloem; VS:

35 vascular tissues; XY: xylem. Scale bars: 150 µm (A); 80 µm (B); 50 µm (C-E); 20 µm

36 (F).

37 Draft

38 Fig. 4 Photomicrographs of secretory glochids in the post-secretory stage in

39 Brasiliopuntia brasiliensis (A) and Nopalea cochenillifera (B, C). (A) Secretory glochid

40 with basal region filled with cells with dense cytoplasm and median and apical regions

41 with elongated cells with lignified walls. (B) Detail of apical portion of secretory

42 glochid filled with cells exhibiting thick lignified walls and less dense cytoplasm. (C)

43 Cladode portion subjacent to the secretory glochid exhibiting dividing parenchyma cells

44 (arrowheads). AP = apical region; BS = basal region; CW: cell wall; MD = median

45 region; PT: protoplast; VS: vascular tissues. Scale bars: 200 µm (A); 25 µm (B, C).

46

47 Fig. 5 Transmission electron micrographs of epidermal cells of secretory glochids in

48 secretory stage (stage 5) in Nopalea cochenillifera. (A-D) Glochid basal region. (E-G)

49 Glochid apical region. (A) Cells with irregular shape, thin walls, large nucleus, and

50 abundant cytoplasm. (B) Epidermal cell with protruding ends lapping over the

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51 neighboring cells. (C-D) Cell portions with dictyosomes, mitochondria, rough

52 endoplasmic reticulum, plastids, vesicles and polysomes. Observe the loose aspect of

53 the cell wall. (E) Epidermal cell with protruding detached portion and less dense

54 cytoplasm. (F) Outer periclinal cell wall with fenestrae and cuticle with electron-dense

55 ramifications. (G) Electron-dense ramifications in the cuticle strata. CT = cuticle; CW =

56 cell wall; DI = dictyosomes; MI = mitochondria; PL = plastids; RER = rough

57 endoplasmic reticulum; VA = vacuole; VE = vesicles. Scale bars: 5 µm (A); 1 µm (B);

58 0.2 µm (C); 0.1 µm (D, F, G); 2 µm (E).

59

60 Fig. 6 Transmission electron micrographs of parenchyma (A-G) and vascular (H) cells

61 in the basal region of secretory glochids in Nopalea cochenillifera. (A-C, H)

62 Conventional method. (D, E) ZIODraft reaction. (F, G) Thiery method. (A) Cells with

63 irregular contour, abundant cytoplasm and developed nucleus with evident nucleolus.

64 (B) Swollen middle lamella in the corners of parenchyma cells. Observe abundant

65 mitochondria and plastids with dense inclusions in the cytoplasm. (C) Cell portion with

66 plastids containing starch grains, mitochondria and vacuoles with flocculent material.

67 (D-E) Dictyosomes with fenestrae and adjacent vesicles impregnated by products of

68 ZIO reaction. Arrows in D indicate plasmodesmata in the cell wall. (F-G) Positive

69 reaction for polysaccharides seen as electron-dense granules in the cell walls and

70 cytoplasm in F and inside plastids in G. (H) Strand of phloem with sieve tube member,

71 companion cells and parenchyma cells. CC = companion cell; CW = cell wall; DI =

72 dictyosomes; MI = mitochondria; ML = middle lamellae; NU = nucleus; PA=

73 parenchyma cell; PL = plastids; ST = sieve tube member; VA = vacuole; VE = vesicles.

74 Scale bars: 5 µm (A, H); 2 µm (B); 3 µm (C); 0.6 µm (D, E); 0.2 µm (F); 0.3 µm (G).

75

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76 Fig. 7 Transmission electron micrographs of ground cells in secretory glochids in the

77 secretory stage in Nopalea cochenillifera (A-C, E, F) and Brasiliopuntia brasiliensis (D,

78 G-I). (A-D) Median region (parenchyma cells). (E-I) Apical region (immature fibers).

79 (A) Cells with thin walls, large nucleus and dense cytoplasm. The arrows indicate

80 plasmodesmata. (B) Cell portions with moderately thick walls and cytoplasm with

81 several mitochondria. (C) Detail showing plastids with thylakoids. (D) Actin strands in

82 the cytoplasm. (E) Cells with very irregular contour and reduced cytoplasm subjacent to

83 the epidermis. (F-G) Cells with thick walls and reduced cytoplasm in the center of

84 glochids exhibiting hexagonal shape in cross section (F) and fusiform aspect in

85 longitudinal section (G). (H) Plasmodesmata (arrows) connecting ground cells in the

86 center of the glochid. (I) Cell portion with mitochondria, plastids with oil drops,

87 polysomes and actin strands. AC= Draftactin strands; CW = cell wall; EP = epidermis; IS =

88 intercellular space; MI = mitochondria; NU = nucleus; OL = oil drops; PL = plastids;

89 VA = vacuole. Scale bars: 4 µm (A); 0.6 µm (B); 1 µm (C); 0.2 µm (D, H); 7 µm (E); 5

90 µm (F, G); 0.4 µm (I).

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Table 1. Histochemical tests for the secretory glochids from Brasiliopuntia brasiliensis and Nopalea cochenillifera (Opuntioideae,

Cactaceae)

B. brasiliensis N. cochenillifera

Reagent Substance Basal Median Apical Basal Median Apical

region region region region region region

Fehling reagent Reducing suggars + - - + - - Ruthenium red Neutral polysaccharides + Draft+ + + + + PAS Acid polysaccharides + + + + + +

Acridine orange Acid polysaccharides + + + + + +

Bromophenol blue Proteins + + + + + +

Sudan IV Total lipids ------

Ferric chloride Phenolic compounds ------

Lugol reagent Starch grains ------

Note: + = presence; - = absence.

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Table 2. Concentration of sugars in the exudate released by glandular glochids in

Brasiliopuntia brasiliensis and Nopalea cochenillifera (Opuntioideae, Cactaceae)

B. brasiliensis N. cochenillifera

Sugar mg g-1 % mg g-1 %

Sucrose 190 88.37 603.15 90.10

Glucose 15.5 7.20 29.95 4.47

Fructose 9.5 4.42 36.25 5.41

Total 215 669.35

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Table 3. Concentration of amiono acids in the exudate released by glandular glochids in

Brasiliopuntia brasiliensis and Nopalea cochenillifera (Opuntioideae, Cactaceae)

Concentration (pmol/µl) Aminoacids B. brasiliensis N. cochenillifera

Aspartic acid b 36.7 4.4

Serineb 457.5 43.6

Glutamic acid b 46.4 3.2

Glycinb 25.7 1.34

Histidinea 7442.7 271.3

Argininea 42.6 2.7

Threoninea 294.1 29.1

Alanineb 312.5 11.1 Prolineb Draft1106.4 101.8 Tyrosineb 55.5 11

Valinea 305.9 50.5

Methioninea 68.6 7.2

Lysinea 25.8

Isoleucinea 250.2 12.7

Leucinea 16.6

Phenylalaninea 45.3 10

Taurinec 69.5 8.1

β alaninec 62 3.3

γ aminobutyric acidc 378.5 34.8

Total concentration 11026 622.74

Protein amino acids concentration (%) 95.37 92.58

Essential amino acids concentration (%) 76.87 64.24

Non-essential protein amino acids 18.50 28.33 concentration (%)

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Non- protein amino acids concetration (%) 4.62 7.41

Note: a = essential aminoacids; b = non-essential aminoacids; c = non-proteinaceous aminoacids.

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Fig. 1 Areoles of young cladodes in Brasiliopuntia brasiliensis (A, B) and Nopalea cochenillifera (C, D). (A) Areole with reduced leaf, lignified spine, secretory glochids and non-glandular hairs. Observe ants visiting the areole. (B) Secretory glochid with liquid exudate droplet at the apex, non- secretory glochids and non- glandular hairs. (C) Secretory glochid with exudate droplet in the apex. (D) Ant removing exudate from the apex of secretory glochid. GG = secretory glochid; HR = non-glandular hairs; LF = reduced leaf; LS = lignified spine; NG = non- secretory glochid. Scale bars: 1 mm (A, D); 0.2 mm (B); 0.5 mm (C).

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Fig. 2 Photomicrographs of the ontogenesis of secretory glochids in Brasiliopuntia brasiliensis. (A) Initial protuberance consisting of protodermal and ground meristem cells. (B) Dome-shaped primordium of secretory glochid consisting of isodiametric cells. (C) Developing secretory glochid with conical shape consisting of cells with dense cytoplasm. (D-G) Developing secretory glochid with distinct basal, median and apical regions. (E) Basal region with isodiametric cells with dense cytoplasm and large nuclei. (F) Median region with isodiametric epidermal cells and slightly elongated parenchyma cells with dense cytoplasm and large nuclei. (G) Apical region with fusiform cells. Note the detached portion of the lower pole of each epidermal cell. AP = apical region; BS = basal region; MD = median region; PC: procambial strand. Scale bars: 50 µm (A, F, G); 100 µm (B, C, E); 150 µm (D).

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Fig. 3 Photomicrographs (A, C-F) and scanning electron micrography (B) of mature secretory glochids of Nopalea cochenillifera (A-C, F) and Brasiliopuntia brasiliensis (D, E). (A) Secretory glochid with basal, median and apical regions. (B) Glochid surface with fusiform-shaped epidermal cells covered by a smooth cuticle. (C) Apical region with fusiform cells. Observe low density of the cytoplasm in the ground cells and the dense and abundant cytoplasm in the epidermal cells. The lower end of the epidermal cells is detached. (D) Median region with isodiametric epidermal cells with dense cytoplasm and fusiform ground cells with less dense cytoplasm. (E-F) Vascular elements in the basal region of the glochids. Observe the cytoplasmically dense parenchyma cells with developed nucleus with evident nucleolus. DR: druse of calcium oxalate. AP = apical region; BS = basal region; MD = median region; PH: phloem; VS: vascular tissues; XY: xylem. Scale bars: 150 µm (A); 80 µm (B); 50 µm (C-E); 20 µm (F).

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Fig. 4 Photomicrographs of secretory glochids in the post-secretory stage in Brasiliopuntia brasiliensis (A) and Nopalea cochenillifera (B, C). (A) Secretory glochid with basal region filled with cells with dense cytoplasm and median and apical regions with elongated cells with lignified walls. (B) Detail of apical portion of secretory glochid filled with cells exhibiting thick lignified walls and less dense cytoplasm. (C) Cladode portion subjacent to the secretory glochid exhibiting dividing parenchyma cells (arrowheads). AP = apical region; BS = basal region; CW: cell wall; MD = median region; PT: protoplast; VS: vascular tissues. Scale bars: 200 µm (A); 25 µm (B, C).

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Fig. 5 Transmission electron micrographs of epidermal cells of secretory glochids in secretory stage (stage 5) in Nopalea cochenillifera. (A-D) Glochid basal region. (E-G) Glochid apical region. (A) Cells with irregular shape, thin walls, large nucleus, and abundant cytoplasm. (B) Epidermal cell with protruding ends lapping over the neighboring cells. (C-D) Cell portions with dictyosomes, mitochondria, rough endoplasmic reticulum, plastids, vesicles and polysomes. Observe the loose aspect of the cell wall. (E) Epidermal cell with protruding detached portion and less dense cytoplasm. (F) Outer periclinal cell wall with fenestrae and cuticle with electron-dense ramifications. (G) Electron-dense ramifications in the cuticle strata. CT = cuticle; CW = cell wall; DI = dictyosomes; MI = mitochondria; PL = plastids; RER = rough endoplasmic reticulum; VA = vacuole; VE = vesicles. Scale bars: 5 µm (A); 1 µm (B); 0.2 µm (C); 0.1 µm (D, F, G); 2 µm (E).

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Fig. 6 Transmission electron micrographs of parenchyma (A-G) and vascular (H) cells in the basal region of secretory glochids in Nopalea cochenillifera. (A-C, H) Conventional method. (D, E) ZIO reaction. (F, G) Thiery method. (A) Cells with irregular contour, abundant cytoplasm and developed nucleus with evident nucleolus. (B) Swollen middle lamella in the corners of parenchyma cells. Observe abundant mitochondria and plastids with dense inclusions in the cytoplasm. (C) Cell portion with plastids containing starch grains, mitochondria and vacuoles with flocculent material. (D-E) Dictyosomes with fenestrae and adjacent vesicles impregnated by products of ZIO reaction. Arrows in D indicate plasmodesmata in the cell wall. (F-G) Positive reaction for polysaccharides seen as electron-dense granules in the cell walls and cytoplasm in F and inside plastids in G. (H) Strand of phloem with sieve tube member, companion cells and parenchyma cells. CC = companion cell; CW = cell wall; DI = dictyosomes; MI = mitochondria; ML = middle lamellae; NU = nucleus; PA= parenchyma cell; PL = plastids; ST = sieve tube member; VA = vacuole; VE = vesicles. Scale bars: 5 µm (A, H); 2 µm (B); 3 µm (C); 0.6 µm (D, E); 0.2 µm (F); 0.3 µm (G).

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Fig. 7 Transmission electron micrographs of ground cells in secretory glochids in the secretory stage in Nopalea cochenillifera (A-C, E, F) and Brasiliopuntia brasiliensis (D, G-I). (A-D) Median region (parenchyma cells). (E-I) Apical region (immature fibers). (A) Cells with thin walls, large nucleus and dense cytoplasm. The arrows indicate plasmodesmata. (B) Cell portions with moderately thick walls and cytoplasm with several mitochondria. (C) Detail showing plastids with thylakoids. (D) Actin strands in the cytoplasm. (E) Cells with very irregular contour and reduced cytoplasm subjacent to the epidermis. (F-G) Cells with thick walls and reduced cytoplasm in the center of glochids exhibiting hexagonal shape in cross section (F) and fusiform aspect in longitudinal section (G). (H) Plasmodesmata (arrows) connecting ground cells in the center of the glochid. (I) Cell portion with mitochondria, plastids with oil drops, polysomes and actin strands. AC= actin strands; CW = cell wall; EP = epidermis; IS = intercellular space; MI = mitochondria; NU = nucleus; OL = oil drops; PL = plastids; VA = vacuole. Scale bars: 4 µm (A); 0.6 µm (B); 1 µm (C); 0.2 µm (D, H); 7 µm (E); 5 µm (F, G); 0.4 µm (I).

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