Secretory structures in plants: lessons from the Plumbaginaceae on their origin, evolution and roles in stress tolerance

The Plumbaginaceae (non-core Caryophyllales) is a family well known for species adapted to a wide range of arid and saline habitats. Of its salt-tolerant species, at least 45 are in the genus Limonium ; two in each of Aegialitis , Limoniastrum and Myriolimon , and one each in Psylliostachys , Armeria , Ceratostigma , Goniolimon and Plumbago . All the halophytic members of the family have salt glands, which are also common in the closely related Tamaricaceae and Frankeniaceae. The halophytic species of the three families can secrete a range of ions (Na + , K + , Ca 2+ , Mg 2+ , Cl − , HCO 3 − , SO 42- ) and other elements (As, Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn). Salt glands are, however, absent in salt-tolerant members of the sister family Polygonaceae. We describe the structure of the salt glands in the three families and consider whether glands might have arisen as a means to avoid the toxicity of Na + and/or Cl − or to regulate Ca 2+ concentrations within the leaves. We conclude that the establishment of lineages with salt glands took place after the split between the Polygonaceae and its sister group the Plumbaginaceae. in related

The Plumbaginaceae Juss. is a cosmopolitan family of perennial herbs, shrubs or small tress, rarely climbers (Kubitzki, 1993) that is well supported as monophyletic (e.g., Cuenoud et al., 2002;Hilu et al., 2003). The family is included in the Polygonids clade of the Caryophyllales (Figure 1 and Table S1), which comprises carnivorous (Ancistrocladaceae, Drosophyllaceae, Droseraceae, Nepenthaceae) and other non-carnivorous sister taxa (Frankeniaceae, Tamaricaceae, Plumbaginaceae and Polygonaceae). The Plumbaginaceae is comprised of two subfamilies, the Plumbaginoideae and the Limonoideae. Genera of the Limonoideae are thought to have initially diversified in the Mediterranean and Irano-Turian regions, although a few genera also occur in the Southern Hemisphere (Lledó, Crespo, Fay, & Chase, 2005;Malekmohammadi, Akhani, & Borsch, 2017;Moharrek et al., 2019;Table S2): Aegialitis is the only genus of the Limonioideae with a tropical distribution (two mangrove species in Asia and Oceania). Within the Plumbaginoideae, its members predominantly occur in arid and saline environments and often in coastal habitats (Hernandez-Ledesma et al., 2015;Kubitzki, 1993;Malekmohammadi et al., 2017;Moharrek et al., 2019). Of the nearly 940 species in the family as a whole, 5% display salt tolerance (Table S2), ranking the family fourth in a list based on the proportion of species within a family that are halophytes (Santos, Al-Azzawi, Aronson, & Flowers, 2016).
Halophytes are plants that survive in the presence of significant concentrations of soluble salt in the medium in which they grow. Quite what amounts to a 'significant concentration of salt' has been a matter of debate over the years (see, e.g., Breckle, 2002;Flowers & Colmer, 2008;Huchzermeyer & Flowers, 2013) with Aronson (1989) selecting a salt concentration of around 80 mM as the dividing line that separates halophytes from more salt-sensitive species, commonly called glycophytes. Flowers and Colmer (2008) used a higher salt concentration, of 200 mM, in order to discriminate higher levels of tolerance.

| MUCILAGE SECRETING GLANDS
The secretions from plants can be produced from both above and below ground organs for a variety of purposes; secretory structures are found in all angiosperm clades (Brown, George, Neugebauer, & White, 2017), with the potential to influence plant microenvironments in various ways (Galloway, Knox, & Krause, 2020). Some plant species have trichomes (called colleters by Fahn, 1979) that secrete mucilage; examples are seen in young stipules of Rumex and Rheum (Polygonaceae). The most common colleters have a stalk with at least two rows of cells, side by side (biseriate), supporting several radiate elongated cells (Fahn, 1979).
Within the Plumbaginaceae, mucilage glands (see Figure 2) were described, in the nineteenth century, in Aegialitis, Armeria, Ceratostigma, Limoniastrum, Limonium and Plumbago in comprehensive studies by Wilson (1890) andde Fraine (1916). These mucilage-glands appear in the axils of leaves and on other organs of all the genera, where they are relatively similar in appearance. In some Limonium species, these glands occur at the base of the leaf sheath on its upper (adaxial) surface (Batanouny, Hassan, & Fahmy, 1992;de Fraine, 1916;Wilson, 1890;Figure 2a), and secrete large quantities of a transparent colourless, viscous liquid at the base of the petioles. The secreting cells are prismatic, columnar or conical and radiate from basal collecting cells with straight periclinal walls, without pores in the cuticle envelope covering the gland cells (Batanouny et al., 1992;de Fraine, 1916;Wilson, 1890).
The mucilages may accumulate either at the cell-wall level or in the space between cell wall and protoplast (Trachtenberg & Fahn, 1981) as well as in vacuoles of epidermal cells (Fahn, 1988). A mucilage histochemical test, tannic acid and iron trichloride (Pizzolato & Lillie, 1973), performed on the lower epidermis of Limonium multiflorum leaves, demonstrates the presence of mucilage (non-structural polysaccharides) by the appearance of a black colour at the cell-wall level and inside a few epidermal vacuoles (Figure 2b). In Armeria, Ceratostigma and Limoniastrum the mucilage glands are of the same type as in Limonium, but in Armeria the basal cells are comparatively few in number, but larger than in species of Limoniastrum (Wilson, 1890). In Aegialitis, the mucilage-secreting cells are found in the axils of the leaves, on laminae, bracts, and sepals, and are very numerous, lying in an oval or circular depression, bounded by regularly-arranged cells (Wilson, 1890). Plumbago species have glandular hairs that secrete a sticky mucilage on the petiole and calyx of flowers (Singh, Naidoo, Bharuth, & Baijnath, 2019;Sudhakaran, 2019;Wilson, 1890). It has been hypothesized that sticky exudates function as an aid to pollination by acting as a barrier for insect predators like ants, so preventing predatory attacks on favoured flying insect pollinators (Panicker & Haridasan, 2016). The calyx glands of noncarnivorous Plumbago are anatomically similar to the mucilage glands of carnivorous genera Drosera and Drosophyllum, suggesting a common ancestral gland structure (Thorogood, Bauer, & Hiscock, 2018).
In carnivorous plants, specific multicellular glands are associated with leaves that have been modified to capture prey. Some of the glands producing secretions are supplied with special vascular strands and the surrounding cells show numerous cell wall plasmodesmata (Guo, Yuan, Liu, & Zhu, 2013;Sharifi-Rad et al., 2017), which regulate the transport of substances between adjacent cells. Many internal secretory structures, like glands and ducts of Euphorbiaceae, Papaveraceae, Clusiaceae and Cannabaceae, are associated with vascular bundles, since compound synthesis requires a regular supply of precursors through the phloem. Plastids and photosynthesis itself are known to be involved in the synthetic pathways of many of the compounds secreted (Evans, 2009) and so a localisation near phloem seems to favour the delivery of these different compounds (Sharifi-Rad et al., 2017). However, the presence of vasculature is not an indicator of functional carnivory, since many glands of carnivorous species are not vascularized (e.g., glands of Nepenthes; Renner & Specht, 2013). Apart from glands secreting mucilage, other glands have evolved without vascular connections, glands that secrete salt (Tables 1 and 2).

| SALT GLANDS
Unlike glands that secrete mucilage, salt-secreting glands are relatively uncommon: they are found in just 12 of the 111 families that contain halophytes. Of the 12 families with salt-secreting halophytes (recretohalophytes), five families contain approximately 90% of the species (Plumbaginaceae, 28%; Poaceae, 21% Amaranthaceae, 20%; Tamaricaceae, 15% and Frankeniaceae, 6%) with seven families containing the remaining 10% (analysis of data in eHALOPH 30/Oct/2019). In all cases, salt glands are epidermal structures, but with anatomical and structural dissimilarities that point to their multiple evolutionary origin . Multicellular salt glands have been described in nine genera of the Plumbaginaceae (Table 1); on leaves and stems, as well as other aerial organs, such as rachis, scapes (inflorescences) and spikes (Salama et al., 1999;Wilson, 1890). Amongst the halophytic species in the family it is likely that all utilize salt glands (see Table 1A), although there are some that thrive in saline habitats/environments, but where the presence of functional glands has yet to be established (e.g., Myriolimon diffusum; Table 1). There are also species with glands, whose salt tolerance has yet to be established; for example, Plumbago zeylanica (Sudhakaran, 2019) and P. europaea (Waisel, 1972) (Thomson et al., 1988); only the glands of Limoniastrum guyonianum (with 32 cells; Tables 1 and 2) and those of Aegialitis (24 or 40 cells) have more. Multicellular salt glands have also been described in the noncore Caryophyllales families Frankeniaceae and Tamaricaceae (Dassanayake & Larkin, 2017;Fahn, 1988;Flowers et al., 2010;Grigore & Toma, 2017;Thomson et al., 1988) -in four genera and 58 species (Table 3; Table S4). Although the number of halophytes within the Frankeniaceae and Tamaricaceae is small, just 2 and 1%, respectively, of all halophytes, 80% of salt-tolerant species within the Frankeniaceae and 67% of salt-tolerant species within the Tamaricaceae have been recorded as having salt glands (eHALOPH, October 2019). Glands within the Frankeniaceae and Tamaricaceae have fewer cells, than genera in the Plumbaginaceae -generally eight (Table S4), rather than 16 in the species within the Plumbaginaceae (Tables 1, 2 and 3). This smaller number of cells per gland (8) is associated with a higher frequency (median of 31 per mm 2 ; n = 14 within the Frankeniaceae and Tamaricaceae, Table S4) than seen in the Plumbaginaceae (median of 12 per mm 2 , n = 22, Tables 1, and S4). Notably, in members of the Polygonaceae, sister group of Plumbaginaceae, no species is recorded as having salt glands.
F I G U R E 2 Limonium mucilage glands. (a). Schematic representation of the cellular organisation of mucilage glands based on de Fraine (1916). Drawing made by Teresa Cardoso Ferreira. (b). Light microscopy of Limonium multiflorum lower leaf epidermis. A histochemical test for mucilage (tannic acid and iron trichloride; Pizzolato & Lillie, 1973), reveals a black colour at both the cell wall (small arrows) and vacuoles (large arrow), demonstrating the presence of those non-structural polysaccharides [Colour figure can be viewed at wileyonlinelibrary.com] T A B L E 1 Genera and species within the Plumbaginaceae (from Plants Of the World Online, POWO) reported to have salt glands (from eHALOPH), their salt tolerance as indicated by inclusion in the database eHALOPH, the position of the glands on the leaf, their frequency and cellular makeup, together with the main elements secreted and rates of efflux The presence of glands is recorded with a ✓ in column 2; a ? indicates the presence of functional salt glands is uncertain or not known. Entries in bold text relate to genera, rather than individual species.
In L. sinense and L. franchetii the mature salt gland is described as a complex structure with 20 cells -four secreting cells each accompanied by an adjacent cell and bounded by four internal and four accessory cells, and four collecting cells (Xin et al., 2011(Xin et al., , 2012 Myricaria Desv. 14 0 3 Reaumuria L. 25 5 3 Tamarix L. 72 29 23 Note: For more detail see Table S4. basal collecting cell and two accessory cells, and these surround a secretory cell (Figure 4a).
The glandular complex can vary in its position in the epidermis.
In leaves, the glands can be seen on the surface at the level of the other epidermal cells or deeply sunken in the leaves, being then side by side with the mesophyll cells (Tables 1 and 2, Figures 4a,b; Akhani et al., 2013;Salama et al., 1999;Thomson et al., 1988). In stems, scapes and spikelets, the insertion of glands is similar to that in the leaves: glands may protrude from the surface as observed in A. maritima (Bernard & Lefebvre, 2001) or even be located on the top of a special elevated cortical structure as reported in Limonium pruinosum (Salama et al., 1999).  (Table S4).

| Physiology of salt secretion
In the late nineteenth century, the secretion of CaCO 3 by glands on the surface of leaves of Armeria, Statice, Goniolimon, Limoniastrum and Plumbago was demonstrated (Braconnot, 1836 (Flowers, 1985;Reef & Lovelock, 2015). The apparent "low exclusion" F I G U R E 4 Legend on next page. can be accounted for by the fact that many halophytes utilize Na and Cl in growth so that xylem concentrations of Na + can be high (see, e.g., Yeo & Flowers, 1986). However, once the delivery of Na + and Cl − exceeds the capacity for the plant to compartmentalize those ions, then growth is reduced. This situation might be avoided or mitigated if excess ions are excreted, but there is too little evidence to allow a clear conclusion. Rozema et al. (1981) showed that excretion by glands can be an important aspect of balancing the salt load in shoots of some species. For plants growing in 200 mM NaCl for 3 days, they (Rozema et al., 1981)  were not particularly reliant on secretion, although even a small proportion could be critical for survival. In a study of mangroves, Reef and Lovelock (2015) concluded that "… salt excretion (the salt gland trait) is not sufficient or necessary to confer high levels of salinity tolerance. The presence of salt glands … is also not linked to levels of salt exclusion." Unfortunately, there is no systematic data for halophytes that compares secretion as a proportion of uptake or the xylem concentrations of Na + and or Cl − that would allow us to evaluate whether or not glands are required for salt tolerance in some species.
As salt tolerant species in the Plumbaginaceae appear to utilize salt glands and yet there is a significant number of species with glands that are not halophytes (Tables 2B, 3 but also through its effects on cell walls and membranes (Greenway & Munns, 1980;Hadi & Karimi, 2012). Ca is essential for plant growth ) and yet the concentration of cytoplasmic free Ca 2+ is low (sub-micromolar, Broadley et al., 2003;Tang & Luan, 2017), reflecting its role as an important signalling molecule. Since there is a strong inward driving force for Ca 2+ into cells (Demidchik, Shabala, Isayenkov, Cuin, & Pottosin, 2018), plants use a variety of means to regulate their Ca concentrations (Tang & Luan, 2017), amongst which is the ability to precipitate calcium oxalate (Franceschi & Nakata, 2005), seen particularly in the Amaranthaceae and Polygonaceae within the Caryophyllales . As far as we are aware, however, little is known of the Ca relations of the Plumbaginaceae, Frankeniaceae, or Tamaricaceae, although shoot Ca concentrations in the Plumbaginaceae and Tamaricaceae, appear to be in the lower range of values seen across plants . The range of Ca concentrations seen in plants is 0.11 to 4.41% of shoot dry weight as calculated by Broadley et al. (2003) from data in the literature. The value for the shoots of Tamarix ramosissima is in the middle of this range, at 1.97% of the dry weight. In a hydroponic experiment they  recorded the shoot Ca of A. maritima to be 0.59%. Since Ca is secreted from the glands of both T. ramosissima and A. maritima (Tables 2 and S4), this is a means by which shoot Ca concentrations could be regulated. We hypothesize that multicellular salt glands could have evolved in the Plumbaginaceae, Frankeniaceae and Tamaricaceae to regulate shoot Ca concentrations and perhaps the balance between Ca and Mg (Tang & Luan, 2017). Over the course of time, this allowed species of these families to colonize drier saline soils as well as seawater (Aegialitis; Table 1).
The gland character is found in species ranging from perennial herbs F I G U R E 4 Salt glands in leaves of Limonium species: a, light microscopy; b and c, fluorescence microscopy; d, e and f, scanning electronic microscopy (plant material fixed in a 2.5% glutaraldehyde solution in 0.1 M sodium phosphate buffer, pH 7.2, for 5 h at 4 C following Hayat (1981). The material was dehydrated in a graded ethanol series (30, 50, 75 to 100% ethanol for 30 min each). Then, leaves were dried on a Critical Point Polaron BioRad E3500 and coated with a thin layer of gold on a Jeol JFC-1200. Observations were carried out at 15 kV on a JSM-5220 LV scanning electron microscope equipped with a direct image acquisition system). (a) Limonium narbonense paraffin leaf cross section cut on a Minot microtome as described in and Ruzin (1999) with slightly fleshy leaves as in Limonium, to cushion-forming dwarf shrubs as in Armeria, coastal shrubs as in Limoniastrum and small trees as in Aegialitis (Table 2), suggesting a plesiomorphic origin of this halophytic trait. Genetic studies in A. maritima provide strong evidence that metallicolous populations have been derived from the ancestral nonmetallicolous populations repeatedly and independently in different geographical regions (Baumbach & Hellwig, 2007).
The evolutionary pathways leading to the "salt glands syndrome" are medium as well as the absorption of water vapour at night (Juniper, Robins, & Joel, 1989;Renner & Specht, 2013 (Baker, 1948(Baker, , 1953Skvarla & Nowicke, 1976;Weber-El Ghobary, 1984). Hence, estimating a divergence date using fossil pollen is not feasible. Based on molecular dating, it has been estimated that the split between the Polygonaceae and its sister group the Plumbaginaceae is relatively ancient, occurring about 90.7-125.0 Ma (million years ago, Schuster, Setaro, & Kron, 2013). Bell, Soltis, and Soltis (2010) (Caperta et al., 2017). Whether salt glands evolved as an ecophysiological innovation to regulate Ca concentrations (discussed above) and adapted to secrete a range of ions (see Tables 2 and 3) including those in saline environments is yet to be clarified. In Armeria, secretory glands are present both in halophytes and metalophytes found in non-saline sandy soils (e.g., A. vulgaris, synonym A. maritima; Baumbach & Hellwig, 2007;Ruhland, 1915) and in sub-mountain meadows and mountain pastures (e.g., A. canescens; Scassellati et al., 2016). Inland populations of A. maritima from sandy and heavy metal soils are salt-tolerant even if less resistant to irrigation with saline water (NaCl) than salt marsh populations (Köhl, 1997).
In the related Tamaricaceae, both non-halophytes such as Myricaria germanica (Dörken, Parsons, & Marshall, 2017; Table S2). Chronogram represents the maximum clade credibility tree estimated in BEAST, with mean divergence dates in million years ago shown for key nodes. Blue bars represent 95% highest posterior density credibility intervals for nodes ages. Values above branches are Posterior Probability. Halophytes (H) and saline glands (G -with glands/nG -without glands), whenever present, information in given for the genus, based on data collected in the bibliographic records (Tables 1-3). Based on the Akaike information criterion calculated in Mega 5.04 (Tamura et al., 2011), general time reversible, GTR + G model of sequence evolution had the best fit to the data. Estimation of phylogenetic relationships and divergence time was conducted using a Bayesian method implemented in BEAST 1.10.4, , employing a strict clock model. Along with the GTR + G model of sequence evolution, we used four rate heterogeneity categories and a Calibrated Yule process for speciation model. We calibrated the root node of our tree, using a normal distribution prior with median = 25 Mya and standard deviation (SD) = 0.5 Mya that covers 95% high posterior probability (HPD). A Marcov chain Monte Carlo analysis was run for 1,000,000 generations and sampled every 1,000 generations. Tracer v1.7, (Rambaut, Drummond, Xie, Baele, & Suchard, 2018), was used to assess that Effective Sample Size (ESS) were about 200 for optimal convergence and tree likelihood stationarity. A maximum clade credibility (MCC) tree was constructed in TreeAnotator v.10.4, , and the MCC Tree visualized in FigTree v1.4, (http://tree.bio.ed.ac.uk/) [Colour figure can be viewed at wileyonlinelibrary.com] or along the branch leading to the Polygonaceae (Schuster et al., 2013) and the branch leading to carnivorous Droseraceae (Rivadavia, Kondo, Kato, & Hasebe, 2003). A phylogenomic sampling based on transcriptomes, show the propensity of polyploidy throughout the evolutionary history of Caryophyllales, and within the non-core Caryophyllales, at least six paleopolyploidy events were inferred . However, direct connections between genome duplication and biological innovations in the Plumbaginaceae are not yet known.

| CONCLUSIONS
The secretory structures that have evolved on the aerial parts of plants can be differentiated between those that secrete organic compounds and those that secrete inorganic ions. All these glands are multicellular structures in dicotyledonous plants, but their ancestry is uncertain. Although the structure of the glands that secrete organic materials is distinct from that of salt glands, it is unclear whether the former gave rise to the latter or if they arose independently. However, salt glands are less common than mucilage glands across the families Aegialitis and Limoniastrum, they evolved independently as they form non-sister groups (Figure 3). In the related Frankeniaceae and Tamaricaceae, families where salt tolerance is closely associated with the presence of salt glands, these structures are composed of just eight cells. The absence of a fossil record means that we cannot evaluate the possibility of a common ancestor of the Plumbaginaceae (16-celled glands) and related families of halophytes like non-core Caryophyllales, Frankeniaceae and Tamaricaceae (8-celled glands).
The prevalence of salt glands in halophytic members of the Plumbaginaceae, Tamaricaceae and Frankeniaceae and the absence of salt glands in the related Polygonaceae, a family with few halophytes, suggest the evolution of these structures is an aid to salt tolerance.
Salt-secreting glands seem to allow the successful colonisation of saline habitats albeit maintained in species thriving in non-saline environments, reflecting the evolutionary independence of this halophytic trait. The presence of such glands also appears to have enabled the colonisation of soils high in heavy metals.