Anatomical and histochemical evidence of leaf salt glands in Jacquinia armillaris Jacq. (Primulaceae)

Journal Pre-proof Anatomical and histochemical evidence of leaf salt glands in Jacquinia armillaris Jacq. (Primulaceae) Vin´ıcius Coelho Kuster, Luzimar Campos da Silva, Renata Maria Strozi Alves Meira

PII:

S0367-2530(19)30497-9

DOI:

https://doi.org/10.1016/j.flora.2019.151493

Reference:

FLORA 151493

To appear in:

Flora

Received Date:

11 January 2019

Revised Date:

20 March 2019

Accepted Date:

24 October 2019

Please cite this article as: Kuster VC, da Silva LC, Meira RMSA, Anatomical and histochemical evidence of leaf salt glands in Jacquinia armillaris Jacq. (Primulaceae), Flora (2019), doi: https://doi.org/10.1016/j.flora.2019.151493

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Anatomical and histochemical evidence of leaf salt glands in Jacquinia armillaris Jacq. (Primulaceae)

Vinícius Coelho Kuster1,2,*, Luzimar Campos da Silva1 and Renata Maria Strozi Alves Meira1

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Universidade Federal de Viçosa (UFV), Departamento de Biologia Vegetal, Av. P.H. Rolfs, s.n., 36570-

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000, Viçosa, Minas Gerais, Brazil. [email protected]; [email protected]. Present address: Universidade Federal de Goiás (UFG), Regional Jataí, Instituto de Biociências, Campus

Cidade Universitária, BR 364, km 195, nº 3800, 75801-615, Jataí, Goiás, Brazil.

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* Corresponding author: [email protected]

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Highlights

The glandular trichomes of J. armillaris correspond to true salt glands.

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The salt glands on J. armillaris are a novelty for the Theophrasteae tribe.

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The salt gland on J. armillaris provides adaptations to coastal environments.

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Abstract Salt glands, which exclude salt solution to avoid tissue toxicity, have been reported for

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halophytes species of Primulaceae. Jacquinia armillaris is a species of Primulaceae that occurs in saline coastal environments in the Neotropics and possesses peltate trichomes

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sunken in leaf surface. The present study investigated these trichomes to describe their structure and check if they are true salt glands. Anatomical and histochemical investigation of the secretory trichomes of the leaves of J. armillaris revealed that they comprise a collecting cell and a single stalk cell with multiple secretory cells located on the top. A collecting chamber originates from the distension and release of the cuticle of the secretory cells. A crystalline substance, which tested positive for sodium, was 1

observed in the collecting chamber. This crystalline salt substance is exuded and deposited on leaf surface. Current results demonstrate that the glandular trichomes of J. armillaris are true salt glands, and thus represent a new record of salt glands for the tribe Theophrasteae of Primulaceae s.l. Keywords: Peltate trichome; halophyte; tribe Theophrasteae; Restinga.

1. Introduction Salt solution covers almost 72 % of the earth’s surface (Flowers and Colmer,

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2008; Flowers and Colmer, 2015), which has resulted in the evolution of complex sets of traits, such as the ability to store Na+ in vacuoles or limiting/restricting the entrance

of sodium ions into the transpiration stream, to salt tolerance by many plants (Flowers et

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al., 2010). Although all plants can tolerate low levels of local salinity, only halophytes

can survive in sites where the salt concentration is equal to or higher than 200-mmol/L

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NaCl (Breckle, 1995). Many halophytes possess salt glands — specialized epidermal

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structures on leaves — that can store and exude salt solution (Dassanayake and Larkin, 2017; Santos et al., 2016) to avoid tissue toxicity. There are almost 370 species of such

Colmer, 2015).

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halophytes (Yuan et al., 2016), which are referred to as recretohalophytes (Flowers and

Salt glands have been reported for about 50 species of 14 angiosperm families of

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Asterids, Ericales, Caryophyllales, Rosids and Grasses (Dassanayake and Larkin, 2017).

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Within Ericales, salt glands have been reported for species of different subfamilies of Primulaceae, such as Aegiceras corniculatum (Cardale and Field, 1971) and Glaux maritima (Rozema et al., 1997) of the subfamily Myrsinoideae (APG IV, 2016), and Samolus repens (Adam and Wiecek, 1983) of the subfamily Theophrastoideae (APG IV, 2016).

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Jacquinia armillaris Jacq. (Primulaceae), of the tribe Theophrasteae, subfamily Theophrastoideae (MOBOT, 2018), occurs in Neotropical dune vegetation (restinga) along the southeastern coast of South America (Castelo and Braga, 2017). The species possesses peltate trichomes sunken in the epidermis of its leaves (Kuster et al., 2018; Luna et al., 2017). These trichomes are presumed to be secretory, but their secretory activities remain unknown, as well as what type of secretory trichomes they are. Halophytes have been reported from coastal sandy environments (Crawford

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2008), including those of Neotropical restinga, which are known to possess high levels of salinity (Scarano, 2002). Plants of restinga environments are also subjected to other adverse environmental conditions, including elevated temperatures (Oliveira et al.,

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2014), coastal flooding (Crawford, 2008; Scarano, 2002), and nutrient poor soils

(Scarano, 2002). Plants that live on the seashore are also influenced by the direct action

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of waves, salty sea spray and high soil salinity (Amaral et al., 2016; Kuster et al., 2018).

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These stressful conditions led Kuster et al. (2016) to test the hypothesis that species occupying these environments should possess salt glands, by histochemically analyzing Ipomoea pes-caprae and I. imperati failed to demonstrate exudation of salt solution.

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The low frequency of salt glands among restinga plants is in agreement with Dassanayake and Larkin (2017), who reported that only a small percentage of

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halophytes possess salt-secreting glands.

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Considering that salt glands have been reported for some species of the family Primulaceae; that the typical environment of J. armillaris is highly saline; and that the species possess anatomical features of secretory trichomes on its leaves, the present study aimed to describe and determine if these trichomes are true salt glands.

2. Material and methods

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2.1. Study area and plant material – The Jacquinia armillaris (Primulaceae) occurs in post-beach formations along the coast of southeastern Brazil. Individual specimens (n=5) were collected from “Paulo César Vinha” State Park in the municipality of Guarapari, state of Espírito Santo, Brazil (23°33’-20°38’S, 40°26’-40°26’W). The species is evergreen and has a scrub habit (~ 2 meters). Voucher specimens were deposited in the herbarium of the Universidade Federal do Espírito Santo (VIES), under

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the number 17,388.

2.2. Anatomical analysis- Mature leaves from the third node (n=6) were collected, fixed in FAA (formalin, acetic acid, 50 % ethanol, 1:1:18 by volume) and stored in 70 %

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ethanol (Johansen, 1940). Leaf samples were embedded in 2-hydroxyethyl methacrylate (Historesin, Leica Instruments, Germany), and sectioned at 5-7 m thickness using a

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rotary microtome (RM2155 Leica, Deerfield, USA). The sections were stained with

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0.05% toluidine blue O (pH 4.0) (O’Brien et al., 1964), and mounted on slides using synthetic resin (Permount, Fisher Scientific, New Jersey, USA). For frontal views of the trichomes, leaf fragments were cleared with 10 % sodium hydroxide solution and 10 %

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chloral hydrate (Johansen, 1940), and then stained with 1% safranin and astra blue solution (Bukatsch, 1972). The slides were mounted in Kaiser’s jelly glycerin (Kraus

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and Arduin, 1997).

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For histochemical analysis, branches with mature leaves were immersed inside containers with ocean water during fieldwork to maintain saline stress. Once in the laboratory, fresh leaves from the third node of the branches were sectioned using a LPC table microtome (Rolemberg & Bhering Trade and Import LTDA, Belo Horizonte, Brazil). Glandular trichomes were tested for primary and secondary metabolites, and for sodium accumulation (Table 1). Control tests were conducted and the slides mounted in

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Kaiser’s jelly glycerin (Kraus and Arduin, 1997) or with the reagent itself, according to the methodology applied (see Table 1). A photomicroscope (AX-70TRF, Olympus Optical, Tokyo, Japan) equipped with an U-photo system (Spot Insightcolour 3.2.0, Diagnostic Instruments Inc., New York, USA) was used to analyze and photograph the material to record anatomical and histochemical data. For epifluorescence microscopy, a HBO50 W mercury vapor lamp and a UV light filter were used. Scanning electron microscopy was performed on both sides of mature leaf

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lamina after fixation and storage, as previously described. The middle region of the leaf was dehydrated in an ethanol series (80% - 90% - 100%) and critical-point dried using liquid CO2 with a CPD 030 Critical Point Dryer (Bal-Tec). The samples were fixed on

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stubs, and covered with gold by a FDU010 sputter coater (Bal-Tec). All samples were photographed using a Zeiss LEO 1430 VP scanning electron microscope (Zeiss,

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Universidade Federal de Viçosa.

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Cambridge, England) at the Núcleo de Microscopia e Microanálises (NMM) of the

3. Results

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3.1. Leaf structure in cross-section– The epidermis is single layered and bears one layer of hypodermis (Figure 1A). The leaf is hypostomatic with stomata at the same level of

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the abaxial epidermal cells (Figure 1A). Multicellular peltate trichomes are present in

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epidermal depressions on both sides of leaf lamina (Figure 1A), and were found intact (Figure 1A, B, C) or already degraded (Figure 1D). The mesophyll is dorsiventral with collateral vascular bundles without vascular sheaths (Figure 1A). Strands of fibers were also observed throughout the mesophyll, some of which were close to the hypodermis (Figure 1A).

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3.2. Description of the glandular trichome and salt exudation – The trichomes comprise a single large basal collection cell with a thin primary cell wall, hyaline cytoplasm, a large vacuole and a peripheral nucleus (Figures 2, 3A). A single cell forms the stalk, which has a thickened cuticle and wall on its lateral side (Figures 2, 3B), dense cytoplasm and evident nucleus (Figures 2, 3A). Cap cells are located on the upper part of the stalk and constitute the secretory portion of the trichome (Figure 2, 3A). The secretory cap is composed of four radially arranged cells, which have thin primary walls

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and thin striated cuticle, as observed in frontal view (Figure 3C). Calcium oxalate crystals are sometimes present in the cap cells (Figure 3D). The cuticle covering the cap cells was frequently distended and detached from the cell wall forming a collection

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chamber (Figures 2, 3A, E).

The structural analysis revealed that the basal and stalk cells possess dense

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cytoplasm and large nuclei when the cap cells are intact, indicating that they have a

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functional role in the collection of salt and its transport to the upper portion of the trichome (Figure 2). The distension and release of the cuticle from the outer periclinal wall was accompanied by the accumulation of saline solution in the chamber (Figure 2,

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3A, E). The subsequent rupture of the cuticle results in exudation, which is followed by the degradation of the trichome cap cells (Figure 1D). The basal and stalk cells become

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vacuolated after saline exudation. The lateral thickened wall of the stalk cell does not

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allow apoplastic transport, thus forcing simplastic transport from inside to the outside.

3.3. Histochemical features of the glandular trichome - No lipid compounds were found between the basal cell and the stalk cell, where a strong reaction for protein occurred (Figure 3F). Wilson’s reagent also revealed the presence of gallic acid derivative at the cell wall between the basal cell and the stalk cell (Figure 3G). No lignin was detected in

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any part of the trichome, demonstrating the pectinaceous nature of the cell walls (Figure 3H). Autofluorescence revealed a crystalline substance in the collection chamber (Figure 3I), which had a positive reaction for sodium (Figure 3J). All other tests were negative for the cap cells and the contents of the collection chamber.

3.4. Leaf surface features - Scanning electron microscopy revealed lifting of the ordinary epidermal cells that surround the trichome, resulting in a depression on each

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side of the leaf (Figure 4A-C). Salt with crystalline appearance is exuded by the peltate trichomes and is accumulated over the depression, on adjacent epidermal cells and over the stomata on the abaxial surface (Figure 4C). The salt is exuded when the cuticle of

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the collecting chamber ruptures, thus eliminating the stored saline content (Figure 4C).

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4. Discussion

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Anatomical features and positive tests for sodium in the collecting chamber of trichomes demonstrated that the glandular trichomes of Jacquinia armillaris are in fact salt glands. These findings represent a new record of salt glands for the tribe

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Theophrasteae of Primulaceae s.l.

Salt glands have been previously reported for halophyte species of the

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subfamilies Myrsinoideae or Theophrastoideae of Primulaceae (Adam and Wiecek,

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1983; Cardale and Field, 1971; Rozema et al., 1977). The subfamily Theophrastoideae contains two tribes: (i) Samoleae, and (ii) Theophrasteae (MOBOT, 2018), without previously reporting of salt glands for the tribe Theophrasteae. Only the genera Jacquinia L. and Clavija Ruiz & Pav belong to Theophrasteae, whose species occur in Central and South America (MOBOT, 2018).

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The salt gland described for the Primulaceae was anatomically referred to as of the standard type (Dassanayake and Larkin, 2017). As observed on the leaves of J. armillaris in the present study, many species of Primulaceae possess multicellular salt glands. However, the features of the salt gland of J. armillaris are more similar to the type described for species of Oleaceae than of Primulaceae (Dassanayake and Larkin, 2017). The salt glands of both species of Oleaceae and J. armillaris have one-collecting cell, one stalk cell, and multiple secretory cap cells (Dassanayake and Larkin, 2017).

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The depression where the salt glands of J. armillaris are located does not seem to be a general characteristic for Primulaceae, but it has been consider a common feature for species of Combretaceae (Dassanayake and Larkin, 2017).

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Jacquinia armillaris is a typical species of restinga, an environment with high

levels of Na+ in the soil (almost 100 mg/dm3 – high according to Alvarez et al., 1999 -

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Kuster et al., 2018). Such high soil salinity has been reported for all restinga

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phytophysiognomies (Crawford, 2008; Kuster et al., 2018; Magnago et al., 2012; Scarano, 2002), and is associated with different anatomical traits such as tolerance mechanisms for cellular toxicity. Water storage tissue (Nguyen et al., 2017; Oliveira et

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al., 2018) and salt glands (Garcia et al., 2017; Munns and Tester, 2008) has been reported for some plants from coastal and saline environments. For instance, leaf, steam

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and/or root of Blutaparon portulacoides (Amaranthaceae) and Remirea maritima

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(Cyperaceae), possess water storage tissue as a mechanism for tolerating high salt concentration (Arruda et al., 2009; Kuster et al., 2018). However, the epidermis is one of the most plastic plant tissues since it is the outermost layer and interacts with the environment, and so exhibits many functional specializations against high salinity at the cellular level (Dassanayake and Larkin, 2017).

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Salt glands are one of the secretory structures of plants that provide competitive success to environmental conditions with high salinity by, in this specific case, helping to increase salt tolerance (Flowers et al., 2010). Salt glands are common in mangrove plants, especially salt-avoiding species, such as those of the genera Avicennia and Laguncularia (Fanh, 1979). Salt glands have been described for some species of coastal and/or saline environments in Australia (Cardale and Field, 1971), United States of America (Levering and Thomson, 1971), China (Feng et al., 2014) and Germany

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(Dörken et al., 2017). However, there are few descriptions of salt glands for plants of Neotropical coastal environments (Kuster et al., 2016). Thus, J. armillaris represents one of the few plants of the restinga known to bear salt glands.

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The anatomical structure of the salt glands of Jacquinia armillares is similar to

that reported for salt glands of other species, including the presence of a depression and

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one collection cell and multiple secretory cells (see Adam and Wiecek, 1983; Gravano

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et al., 2008). Moreover, salt crystals exuded by trichomes have been reported on leaves of plants growing in environments with high salt concentrations, and have been used as an indicator of the occurrence of salt glands in halophytes (Céccoli et al., 2015; Garcia

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et al., 2017; Taleisnik and Anton, 1988). Thus, the presence of salt crystals provides additional support for the characterization of the secretory trichomes of J. armillaris as

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salt glands.

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The presence of calcium oxalate crystals in the secretory cells of salt glands of J. armillaris is similar to what was reported for salt glands of Phillyrea latifolia L. (Oleaceae), which was associated with the maintenance of local ion concentrations (Gravano et al., 2008). The thick cuticle on the lateral walls of the stalk of the trichomes of J. armillaris has been reported for other types of trichomes, such as mucilaginous trichomes of Ipomoea imperati and I. pes-caprae (Kuster et al., 2016), and has been

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associated with the prevention of backflow of substances into the mesophyll (Bosabalidis and Tsekos, 1982; Semenova et al., 2010). The strong positive test for proteins in the cell walls and lipid membranes between the basal collection cell and the stalk of J. armillaris may be due to the presence of proteins that act in sodium transport, such as chloride channel and Na+/H+ antiporters (Yuan et al., 2016). Lastly, Wilson’s reagent has been previously used to evidence the presence of gallic acid by Slaughter et al. (2008), and demonstrated the presence of this compound in the inner periclinal wall

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of the stalk cell of the salt gland of J. armillaris. Gallic acid is a phenolic compound that is involved in antioxidant activity (Balasundram et al., 2006) and the induction of apoptosis (Inoue et al., 2000), but it has not been reported to be involved in the

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functioning of salt glands.

Studies on the process of salt exudation from salt glands over recent decades

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have revealed different mechanisms for Na+ and Cl- exudation (Ma et al., 2011), as well

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as the exudation of ions, such as K+, Ca+2, N-3, Mg+2, among others (Céccoli et al., 2015; Oi et al., 2013; Zouhaier et al., 2015). The penetration of ions occurs in the lower part of salt gland due to fluid pressure generated by plasmodesmata, with salt accumulation

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in the collection chamber and frequent detachment of the cuticle (Yuan et al., 2016). The exudation of salt solution may occur via pores in the cuticle or by its rupture (Yuan

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et al., 2016). For instance, salt glands with cuticle containing pores involved in the

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excretion of saline solution were reported for Avicennia marina (Shimony et al., 1973), Spartina foliosa (Levering and Thomson, 1971) and Myricaria germanica (Dörken et al. 2017). For plant as Aegiceras corniculatum (Cardale and Field, 1971), the presence of pores in the cuticle was not found. On the other hand, in Glaux maritima only a few times the pores were found in the secretory cap (Rozema et al., 1977), which leads to doubts about its effectiveness in the excretion of substances. The detachment and

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rupture of the cuticle for salt elimination were described in the present work for J. armillaris, as reported for the salt glands of Sporobolus virginicus (Naidoo and Naidoo, 1998), however, the ultrastructural study is needed to better understanding of J. armillaris salt exudation. Halophytes have many strategies for tolerating the high levels of salinity of the environments where they occur, with the exudation of salt solution by salt glands being specific to some groups of plants. In this sense, the anatomical and histochemical

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evidence here supports that the peltate trichomes sunken of J. armillaris are true salt glands, and it represents a significant data for understanding ecological responses by

restinga plants and the structural diversity of these secretory structure. Moreover, the

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salt glands of J. armillaris represent a first and new record for the tribe Theophrasteae

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of Primulaceae s.l.

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5. Acknowledgments

The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the Research Productivity Scholarships granted to L.C. Silva

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(309308/2018-6) and R.M.S.A. Meira (308389/2013-1). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil

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(CAPES) - Finance Code 001, which has also granted the scholarship to the first author.

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PS: The color should be used for any figures just for online version.

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Captions

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Figure 1. Anatomical characteristics of the leaf of Jacquinia armillaris. A- Internerval region; B- Presence of salt glands on both sides of the leaf blade; C- Intact salt gland; D- Degraded salt gland. Abbreviations: BC- Basal Cell; CC- Cap Cells; Fi- Fibers; GT-

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Glandular trichome; Hy- Hypodermis; PP- Palisade Parenchyma; SC- Stalk Cell; SP-

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Spongy Parenchyma; St- Stomata; VB- Vascular Bundle.

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Figure 2. Illustration of the salt gland of Jacquinia armillaris.

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Figure 3. Anatomical and histochemical features of salt glands of Jacquinia armillaris. A- Structure of a mature salt gland; B- NADI test showing the thick cuticle on the lateral wall of the stalk cell (arrow); C- Frontal view; D- Druses in cap cells (arrow); E-

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Salt secretion by a salt gland; F- Protein present in the cell wall between basal and stalk cells as indicated by Xylidine ponceau; G- Wilson´s reagent showing gallic acid

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derivate between basal and stalk cells (arrow); H- Pectinaceous nature of the cell wall of

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a salt gland (arrow); I- Autofluorescence showing crystalline substance in the collecting chamber (circle); J- Positive reaction to the Uranyl zinc acetate test indicating sodium in the collecting chamber (circle). Abbreviations: BC- Basal Cell; Ca- Cavity; CC- Cap Cells; CCh- Collecting Chamber; Cu- Cuticle; Fi- Fibers SC- Stalk Cell. Bars = 25 m.

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Figure 4. Scanning electron microscopy of both sides of the leaf of Jacquinia armillaris.

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A- Adaxial suface; B, C- Abaxial surface; C- Salt secretion by a salt gland and its

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accumulation on the leaf surface. Abbreviations: Sa- Salt; SG- Salt Gland; St- Stomata.

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Table 1. Metabolite groups investigated and methodologies adopted in the histochemical study of leaf trichome of Jacquinia armillaris.

General

Lipids

General Sodium

Uranyl zinc acetate

Total lipids

Sudan black B Copper acetate and rubeanic acid Ferric chloride Potassium dichromate Vanillin–hydrochloric acid Phloroglucinol Wilson´s reagent Aluminum chloride Periodic Acid – Schiff´s reagent (PAS) Lugol Ruthenium red

Fatty acids General

Phenolic compounds

Tannins Lignin Flavonoids

Callose

Xylidine ponceau

Essential oils and resin-oils Terpenoids with carbonyl group

Nadi reagent

Steroids

Antimony trichloride

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Terpenoids

Aniline blue

Total proteins

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Proteins

Furr and Mahlberg (1981)

Alcian blue

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Polysaccharides

Neutral polysaccharides Starch Pectins Acid mucopolysaccharides

Authors

Jensen (1962), Harvey (1987) Brundrett et al. (1991) Ganter and Jollés (1969)

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Alkaloids

Inorganic compounds

Reagents Wagner´s reagent Dittmar´s reagent Ellram´s reagent Autofluorescence

Johansen (1940) Gabe (1968)

Mace and Howell (1974)

Johansen (1940) Charrière-Ladreix (1976) Charrière-Ladreix (1976)

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Compounds

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Metabolite groups

2,4dinitrophenylhydrazine

Sesquiterpene lactones

Sulfuric acid

Rubber

Oil red O

McManus (1948) Jensen (1962) Johansen (1940) Pearse (1980) Smith and McCully (1978) O’Brien and McCully (1981) David and Carde (1964) Ganter and Jollés (1969) Hardman and Sofowora (1972), Mace et al. (1974) Geissman and Griffin (1971) Jayabalam and Shah (1986)

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