Dealing with extremes: insights into development and operation of salt bladders and glands

Abstract

Salt bladders and salt glands of recretohalophytes are specific salt-secreting structures evolving from trichomes that allow plants to adapt to extreme environmental conditions (such as salinity or drought) by precise regulation of the salt load in metabolically active leaf tissues. The knowledge of the process by which these structures are formed and operate may provide a blueprint for introducing them into cultivated crops thus creating climate-resilient genotypes. This review summarizes the recent progress in understanding mechanisms involved in the development and operation of salt bladders and glands, at physiological, molecular, and genetic levels. We show that these processes differ depending on the anatomical structure of glands (unicellular vs bi-cellular vs multicellular), as well as between salt glands and bladders. We also show that recretohalophytes with different types of salt glands may have different trichome development. We discuss new evidence for the role of vesicular transport in salt secretion and highlight the importance of multi-omics in understanding mechanisms of salt secretion and sequestration in epidermal bladder cells, and their contribution to salinity stress tolerance in plants.

Keywords:

• Salt tolerance
• epidermal bladder cell
• salt gland
• trichome
• development
• secretion

The importance of secretory structures in salinity tolerance

Soil salinity imposes three major constraints on plants, namely osmotic stress, nutritional disbalance (ion toxicity), and oxidative stress (Zhao et al., 2020). The latter two are manifest in shoots, and their impact can be minimized by the efficient removal of potentially harmful ions (Na⁺ and Cl^-) from metabolically active intracellular compartments. In most cases, this process implies sequestration of excessive harmful ions in vacuoles. Both halophytes and glycophytes use this mechanism, although with different levels of efficiency (Shabala and Mackay, 2011; Flowers and Colmer, 2015). Another way of handling a toxic salt load in the shoots is to remove excessive ions from mesophyll, either by storage in epidermis bladder cells (EBCs) or by transfer to salt glands and microhairs (for secretion). All these structures are classified as modified trichomes (Shabala et al., 2014). They originate from epidermis cells followed by their differentiation into unicellular or multicellular structures (Johnson, 1975; Adams et al., 1998; Szymanski et al., 2000). Most plant species have trichomes on the surface of their leaves; however, only halophyte species possess this ability to deposit the salt into these structures. We have previously argued that understanding the mechanistic basis of EBC and gland formation and operation may allow this trait to be introduced into domesticated crop species (Shabala, 2013; Shabala et al., 2014; Rawat et al., 2022). Although this idea is gaining momentum (Dassanayake and Larkin, 2017), it is still in its infancy, due to a shortage of available high-quality genome data on halophytes, a lack of genetic transformation systems, difficulties in isolating secreting structures, and limited availability of germplasm resources. This review summarizes up-to-date progress in the field focusing on mechanisms involved in the development and operation of salt bladders and glands, at physiological, molecular, and genetic levels.

Types of salt-secreting modified trichomes

Trichomes are accessory structures that originate from plant epidermal cells (Adams et al., 1998; Werker, 2000; Balkunde et al., 2010). In glycophytes, trichomes are typically classified into glandular trichomes (GTs) and nonglandular trichomes (NGTs), depending on their secretory ability (Singh et al., 2019). In recretohalophytes, modified trichomes are categorized as salt glands and salt bladders, based on their morphology. Although they also originate from epidermal cells, they evolve to have functions that differ from those of glycophytes, particularly in salt secretion under saline conditions (Shabala et al., 2014; Yuan and Wang, 2020; Yun and Shabala, 2021; Koteyeva et al., 2022) and, as such, play a vital role in salt tolerance of halophytes (Marcum, 1999; Kiani‐Pouya et al., 2017).

Each salt bladder complex consists of an epidermal bladder cell with a notably large vacuole, and a stalk cell (Figure 1A). EBCs are numerous on both abaxial and adaxial leaf surfaces in Atriplex centralasiatica, Chenopodium quinoa and Mesembryanthemum crystallinum. In a recent study, Lai et al. (2022) reported that Glycine soja, a wild relative of cultivated soybean, possesses salt glands on the leaf surface. However, using the term “gland” is misleading in this context, as no secretory activity was shown and, anatomically, these structures consisted of a large bladder cell and a stalk cell, similar to the salt bladders of Atriplex and Chenopodium. Once the genes conferring these structures in G. soja are known, it may be possible to transfer them to cultivated soybean species, in order to improve the ability to tolerate high salinity in the soil.

Figure 1. Structure of salt-secreting modified trichomes in recretohalophytes. A. A salt bladder complex includes an epidermal bladder cell (EBC) and a stalk cell in C. album (from Zhang et al. (2022) and only EBC in M. crystallinum (from Oh et al. (2015); scalebar: 0.5 mm); B. Salt gland structures include single salt gland in O. coarctata (Koteyeva et al., 2022; n, nucleus; scalebar: 10 µm), bicellular salt gland in Chloris gayana Kunth (Oi et al., 2012; BC, basal cell; BR, base region of basal cell; Ca, cavity; CC, cap cell; Cu, cuticular layer; E, epidermal cell; M, mesophyll cell, n, nucleus; NR, neck region of basal cell; Va, valleculae; arrows: plasmodesmata) and multicellular salt gland in L. bicolor (Yuan et al., 2015; SC, secretory cell; AC, accessory cell; IC, inner cup cell; OC, outer cup cell; CC collection cell; scalebar: 5 µm). Modified from: A - Zhang et al. (2022) and Oh et al. (2015); B – Koteyeva et al. (2022), Oi et al., (2012) and Yuan et al. (2015) with permission from John Wiley and Sons; Springer Nature; and University of Chicago Press.

EBCs secrete salt only once upon their rupture, while, by contrast, salt glands do it on a regular basis. Salt glands can be further categorized into unicellular, bicellular and multicellular (Yuan and Wang, 2020; Yun and Shabala, 2021). Multicellular salt glands are commonly found in dicots such as in mangroves like Avicennia marina (Shimony et al., 1973; Chen et al., 2010; Guo et al., 2023), or Aegiceras corniculatum (Cardale and Field, 1971; Field et al., 1984), as well as in Limonium bicolor (Leng et al., 2018; Zhao et al., 2023), and Tamarix aphylla (Thomson et al., 1969; Bosabalidis, 2012). Bicellular salt glands are only found in monocots, more precisely in the Poaceae such as Zoysia matrella (Rao, 2011; Yamamoto et al., 2016), Chloris gayana (Oi et al., 2012, 2013, 2014), Sporobolus anglicus and Urochondra setulosa (Koteyeva et al., 2022). Here, a basal cell and a cap cell operate in collecting salt and in secretion, respectively (Bicellular salt gland of Figure 1B; Oi et al., 2012; Koteyeva et al., 2022). The wild rice Oryza coarctata is a special example of a halophyte with a single-cell salt gland (Single salt gland of Figure 1B), and salt is secreted through these glands (Koteyeva et al., 2022) but not the co-located microhairs (Flowers et al., 1990; Rajakani et al., 2019). Although multicellular and bicellular salt glands differ with respect to cell number and composition, they all contain collecting cells and secretory cells, and all glands typically comprise a cuticle envelope, cuticular chamber, cap cell, and basal cell (Multicellular salt gland of Figure 1B) Unicellular salt glands operate in a different way that has not been characterized yet.

In addition to salt-secreting structures including EBCs and salt glands found in halophytes, other potentially secreting structures in glycophytes have also been found to secrete salt. For example, microhairs on the surface of leaves of Zea mays have been shown to secrete salt, and the microhair number and secretion rate could increase with increasing salinity concentration (Ramadan and Flowers, 2004). Johnsongrass could discharge salt via trichomes on the surface of its leaves when grown not in soil with high NaCl concentration but rather in soil containing lime (McWhorter et al., 1995). Salt crystals have been found on the leaf surface of Gossypium hirsutum after NaCl treatment, and this phenomenon was more obvious in the salt-tolerant genotype compared to the salt-sensitive one. Glandular trichomes were responsible for the salt secretion (Peng et al., 2016), potentially conferring stronger salt-tolerance to the upland cotton.

How do salt bladders and salt glands form?

Trichome formation is a complex process that may involve several different mechanisms (Serna and Martin, 2006). Most of the current knowledge comes from nonhalophytes and, specifically, Arabidopsis, where transcription factors such as R2R3 MYB, HD-ZIP IV, and basic helix-loop-helix (bHLH) were shown to play a critical role. It was shown that an R2R3 MYB transcription factor GLABRA1 (GL1), a bHLH transcription factor GLABRA3 (GL3) and ENHANCER OF GL3 (EGL3), and a WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) can form a so-called MBW complex to activate the downstream gene HD-ZIP IV GLABRA2 (GL2) for initiating trichome differentiation (Zhang et al., 2003; Serna and Martin, 2006; Morohashi et al., 2007; Zhao et al., 2008). Besides, the MBW complex can activate TRIPTYCHON (TRY), CAPRICE (CPC), ENHANCER OF TRY AND CPC 1 (ETC1), ETC2, ETC3, TRICHOMELESS1 (TCL1) and TCL2, promoting their diffusion to neighboring cells to compete with GL1 for binding to GL3, thus locally suppressing trichome formation (Kirik et al., 2004a, 2004b; Wang et al., 2007; Pesch and Hülskamp, 2009; Edgar et al., 2014; Wang and Chen, 2014). Recently, this regulatory mechanism has also been described in Camellia sinensis where CsMYB1 interacts with CsGL3 and CsWD40 to form a transcriptional MBW complex of MYB-bHLH-WD40 to activate trichome regulator genes CsGL2 and CsCPC and this MBW complex also participates in catechin biosynthesis in tea (Li et al., 2022). These findings suggest that trichome development is a complex process where various transcription factors form a type of complex to interact with other transcription factors, and in plants like C. sinensis this complex could be involved in multiple biological pathways.

Salt bladder development-related genes

Despite EBC being a prominent feature in many species, most of the knowledge comes from M. crystallinum and C. quinoa. A small number of genes involved in EBCs or trichome formation have been partially characterized (Table 1). In M. crystallinum, WM28, a putative jasmonate-induced gene, may elevate GL1 and GL3 expression known to facilitate trichome development in Arabidopsis (Roeurn et al., 2016), indicating this gene could potentially be involved in EBC formation. Homologs of trichome development-involved genes in A. thaliana have been found in both M. crystallinum and C. quinoa and have been reported to participate in EBC development. R2R3-MYB McMYB2, the homolog of AtMYB5, can interact with bHLH proteins and a WD40 protein to form a MBW complex that regulates trichome development. The homolog of GL2, McC4HDZ encoding a Class IV Homeodomain Leucine Zip (HD-Zip IV) protein, can recover the phenotype of the Arabidopsis gl2 mutant to some extent, thus taking over GL2 function in this species. Thus, this gene may contribute to EBCs formation (Roeurn et al., 2017). Imamura et al. (2020) identified a gene, REDUCED EPIDERMAL BLADDER CELLS (REBC), encoding a WD40 protein, which was responsible for EBC formation in quinoa. In a rebc mutant the number of EBCs decreased to less than 0.5% of the wild type. However, REBC has a low similarity to WD40-protein encoded by TTG1 in Arabidopsis and REBC cannot complement the ttg1 mutant of that species, whereas two quinoa homologs of TTG1, CqTTG1-like1 and CqTTG1-like2 can do this. This indicates that CqTTG1-like1/2 may be involved in root hair but not in trichome development because there are no other trichomes on the leaf surface of quinoa. Mutating simultaneously REBC and its homolog, REBC-like1, completely suppressed EBC formation on the leaf surface and on the inflorescence of quinoa plants, which was not observed by only mutating REBC-like1. Hence, REBC-like1 may act as an enhancer of REBC in regulating EBCs formation (Moog et al., 2022). These studies suggest EBC formation is likely to be different from the trichome formation of Arabidopsis. In the future, TTG1 in Arabidopsis needs to be tested with respect to its ability to complement rebc or/and rebc-like1 mutants of quinoa when the genetic transformation system for quinoa becomes mature, to further supplement or refine our knowledge of the mechanisms of EBC formation. Interestingly, rebc rebc-like1 double mutants did not differ from the wild types in terms of salt tolerance when treated with low and high NaCl concentrations, despite the mutants lacking EBCs completely, which contradicts the accepted view that EBCs contribute to salt tolerance of quinoa by Na⁺ and Cl^- secretion (Agarie et al., 2007; Kiani‐Pouya et al., 2017; Kiani-Pouya et al., 2019; Yun and Shabala, 2021). However, it should be noted that in Moog et al. (2022) WT plants responded to salinity treatment by a dose-dependent decline in their biomass (i.e., possessed a glycophytic type of response) while in other papers cited above moderate levels of salt (around 100 mM NaCl for quinoa) were found to be optimal for plant growth. It appears that some other factors (in addition to salinity) affected plant performance in these experiments, making a direct comparison impossible. In lay terms: under conditions of experiments by Moog et al. (2022) WT quinoa plants did not behave like a halophyte and, hence, may not have used EBCs to deal with the salt load.

Table 1. Genes involved in formation of salt bladders or glands in recretohalophytes.

Gene name	Function	Descriptions	Species	Reference
WM28	↑ trichome number in Arabidopsis; ↑ probably EBC development	A putative jasmonate-induced gene	M. crystallinum (Salt bladders)	(Roeurn et al., 2016)
McMYB2		R2R3 MYB transcription factor		(Roeurn et al., 2017)
		HD-ZIP IV transcription factor
McC4HDZ
REBC	↑ EBCs and chloroplasts formation	Encodes a WD40 protein; locates to the nucleus and chloroplasts	C. quinoa (Salt bladders)	(Imamura et al., 2020)
CqTTG1-like1	↑ trichome development	Homologs of TTG1 in Arabidopsis and encode a WD40 protein
CqTTG1-like2
REBC-like1	No effects on EBCs formation when this gene exists only			(Moog et al., 2022)
LbHLH	↑ trichome development; ↓ root hair differentiation in Arabidopsis; ↑ salt gland density and appearance; ↑ salt tolerance of Arabidopsis; ↓ salt tolerance of sea lavender	A bHLH transcription factor but less than 30% sequence similarity with that in Arabidopsis; locates to the nucleus	L. bicolor (Salt glands)	(Wang et al., 2021; Yuan et al., 2022)
LbTTG1		Encodes a WD40-repeat protein with high sequence similarity to TTG1 in Arabidopsis; locates to the nucleus		(Yuan et al., 2019, 2022)
LbSAD2	↑ trichome development; ↓ root hair differentiation in Arabidopsis; ↑ salt tolerance of Arabidopsis	Encodes an importin-β protein		(Xu et al., 2020)
LbMYB48	↑ salt gland development and salt secretion ability; ↑ tolerance of Arabidopsis	R1type MYB transcription factor; locates to the nucleus		(Han et al., 2022)
Lb1G04794	↑ trichome development; ↓ regulate root hair differentiation in Arabidopsis; ↑ salt gland development; ↑ salt tolerance of Arabidopsis	Do not contain transmembrane domain, signal peptide and conserved domain; locates to the nucleus		(Jiao et al., 2022)
LbCPC	↓ salt gland development and density; ↓ trichome number, ↑ root hair number, ↑ salt sensitivity of Arabidopsis	MYB transcription factor; locates to the nucleus		(Zou et al., 2023)

Notes: ↑ and ↓ means positive and negative regulation, respectively.

Salt gland development-related genes

L. bicolor is arguably the most studied species for understanding salt gland development in halophytes, but knowledge of genes involved in salt gland development is very limited (Table 1). Even though homologs of some trichome development-related genes like GL1, TTG1, GL3/EGL3 can be detected in L. bicolor, homologs of GL2 and TTG2 are absent (Table 2), which may explain why there is no other type of trichomes on its leaf surface (Yuan et al., 2022). Remarkably though, GL2, GL3 and TTG2 are highly conserved among glycophytes and other recretohalophytes, respectively, as shown in Figure 2. A homolog of TTG1, LbTTG1 has been detected in L. bicolor, and Yuan et al. (2019) found that when overexpressed heterologously in Arabidopsis, LbTTG1 could up-regulate the expression of GL1 and GL3, promoting trichome development. A decreased expression of TRY and CPC inhibits trichome development, consistent with the function of TTG1 in trichome development in Arabidopsis (Zhao et al., 2008; Yuan et al., 2019). It was also shown that LbHLH functions in the same way as LbTTG1 when interacting with GL1 (Wang et al., 2021). However, these two genes act as a negative regulator in salt gland development, as overexpression of LbTTG1 and LbHLH in L. bicolor led to a reduced salt gland number and abnormal salt glands (Yuan et al., 2022). Meanwhile, LbHLH cannot complement gl3 single and gl3 egl3 double mutants. These reports suggest that genes involved in salt gland development in recretohalophytes like LbTTG1 and LbHLH have specific functions that differ from those in nonhalophytes, although they have the ability to participate in trichome and root hair development to some degree. Interestingly, the homologs of TTG1 are absent in O. coarctata (Table 2), suggesting that such unicellular salt gland development may differ from the bicellular and multicellular salt glands provided that TTG1 really participates in bicellular and multicellular salt gland development. In L. bicolor, R1-type MYB LbMYB48 might indirectly negatively regulate the expression of LbCPC-like, a homolog of AtCPC known to bind TTG1 and GL3 to form complexes inhibiting trichome development in Arabidopsis and DISTORTED3 (LbDIS3) to negatively regulate the formation of salt glands (Wada et al., 1997; Tominaga et al., 2007; Han et al., 2022). Another gene, Lb1G04794, located in the nucleus, promoted salt gland development as a transcription factor when overexpressed in L. bicolor, while also increasing trichome number but reducing root hairs when overexpressed in Arabidopsis (Jiao et al., 2022). Unlike Lb1G04794, a MYB LbCPC, that is homologous to AtCPC, has the opposite effect. This gene inhibited salt gland formation and density when overexpressed in L. bicolor and inhibited trichome development. It was shown that it may combine with GL3 to restrain the formation of TTG1-GL3-GL1 complex when overexpressed in Arabidopsis (Zou et al., 2023). Except for transcription factors, Limonium homologs of other genes in Arabidopsis involved in trichome initiation have been reported. LbSAD2, a homolog of SUPER SENSITIVE TO ABA AND DROUGHT2 (AtSAD2), has been reported to positively affect trichome numbers through mediating probably GL3 and improving salt tolerance when overexpressed in Arabidopsis. This gene was located and highly expressed in the salt gland during their formation of L. bicolor (Xu et al., 2020), indicating its functional role in this process.

Figure 2. Maximum likelihood tree of GL2 (A), TTG2 (B) and GL3 (C) proteins from glycophytes and recretohalophytes. Genes in glycophytes and recretohalophytes were marked by solid circle. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA X.

Table 2. Homologs of trichome development-related genes in glycophytes and recretohalophytes.

Group	Trichomes or modified trichomes	Organism	Absence or presence of genes
			R2R3-MYB	WD40	bHLH	HD-Zip	WRKY	RBR1	SIM	R3-MYB
			GL1	TTG1	GL3/EGL3	GL2	TTG2			TRY	CPC	ETC1/2/3	TCL1/2
Glycophytes	Trichomes	A. thaliana	+	+	+	+	+	+	+	+	+	+	+
		G. hirsutum	+	+	+	+	+	+	+	+	+	+	+
		A. annua	+	+	+	+	+	+	–	+	+	+	+
		C. sativus	+	+	+	+	+	+	–	+	+	+	+
		S. lycopersicum	+	+	+	+	+	+	–	+	+	+	+
		O. sativa	+	+	+	+	+	+	–	+	+	+	+
Recretohalophytes	Unicellular salt glands	O. coarctata	+	–	+	+	+	+	–	+	+	+	–
	Bicellular salt glands	Z. matrella	+	+	+	+	+	+	–	+	+	+	+
		A. littoralis	+	+	+	+	+	–	–	+	+	+	+
	EBCs	C. quinoa	+	+	+	+	+	+	–	+	+	+	+
	Salt glands or EBCs	G. soja	+	+	+	+	+	+	–	+	+	+	+
	Multicellular salt glands	A. corniculatum	+	+	+	+	+	+	–	+	+	+	+
		A. marina	+	+	+	+	+	+	–	+	+	+	+
		L. bicolor	+	+	+	–	–	+	–	+	+	+	+

How do salt bladders or salt glands of recretohalophytes secrete NaCl?

There is currently a limited knowledge on the molecular mechanisms conferring ion transport to salt glands and bladders and their secretion. The major hurdles are difficulties in isolating them, and the absence of a genetic transformation systems. As a result, most of the current knowledge comes from microscopical studies and multiple omics including genomics, transcriptomics, proteomics, metabolomics and ionomics.

The majority of published research originates from salt bladders of C. quinoa and M. crystallinum. As the progress on ion transport of salt bladders has been reviewed recently by Yun and Shabala (2021), it will not be discussed in detail in this section. Instead, we will focus on ion transport and secretion in salt glands. The latter involves three steps: the entry of ions into the glands from mesophyll and epidermal cells; the transport of ions within the salt glands; and the secretion of ions out of the glands. While several hypotheses have been proposed regarding salt secretion in recretohalophytes, it is still unclear which pathways - apoplastic or symplastic pathways or both – play a key role in the transfer of ions from mesophyll and epidermal cells to the basal cells of the glands. Plasmodesmata have been observed between basal cells and neighboring mesophyll and epidermal cells in dicots and the Poaceae S. virginicus, S. anglicus and U. setulose (Naidoo and Naidoo, 2008; Koteyeva et al., 2022). However, plasmodesmata were present only between the gland and adjacent epidermal cells in O. coarctata (Koteyeva et al., 2022), and in other species (Z. matrella) such plasmodesmata were reported for some but not all genotypes (Rao, 2011).

In Z. matrella, ZmatHKT1, Na⁺-K⁺ transporter was predominately expressed in the salt glands, bundle sheath cells and mesophyll cells between salt glands in the adaxial side leaf, but not in the nonsecreting abaxial side (Rao, 2011), indicating ZmatHKT1 would be likely to participate in ion transport into basal cells from their adjacent cells. Once the ions enter the salt glands, they move via similar pathways. Additionally, these ions are likely to enter the secretory cells via vesicle transport. Electron-dense zones were found inside the vesicles by Thomson and Liu (1967), and these vesicles could fuse with the plasma membrane after salt treatment (Feng et al., 2014; Yuan et al., 2015). Using Corona Green, a sodium dye, we localized the Na⁺ distribution and found that Na⁺ was enriched in these vesicles. The salt glands showed more Na⁺ in their vesicles under saline conditions when compared to the control without salt as shown in Figure 3. Hence, ions, at least Na⁺, could be transported from the basal cells to secretory cells by exocytosis. Finally, the ions enter the collecting chamber and are secreted out of the salt glands through pores. Four pores were observed in L. bicolor using scanning electron microscopy (SEM) (Leng et al., 2019) whereas no pores were found on the globular surface of salt glands in the Poaceae recretohalophyte Rhodes grass (Chloris gayana Kunth) using SEM (Oi et al., 2013). This raises the question if ions are secreted via pores or if other pathways are used, calling for more studies on these issues.

Figure 3. Confocal imaging of Na⁺ fluorescence using CoroNa Green AM in salt glands of A. lagopoides under control and 300 mM NaCl for 3 days.

In addition to the aforementioned Na⁺, Cl^- and K⁺ could also be secreted via salt glands alongside a long list of other inorganic and organic compounds such as macro- and micro-elements, toxic metals and even some metabolites (Pollak and Waisel, 1970; Kadukova et al., 2008; Naz et al., 2009; Ding et al., 2010). Glandular trichomes (GTs) can produce, store, and secrete secondary metabolites (Konarska and Łotocka, 2020; Sun et al., 2022). In addition, GTs in the leaves of G. hirsutism have the ability to secrete salt ions (Peng et al., 2016), and GTs of tobacco can secrete cadmium (Cd) and calcium (Ca) (Choi et al., 2001), and secrete more cadmium than NGTs in a Cd-enriched environment (Liu et al., 2019). Salt glands exhibit similar secretion functions, including ions and metabolites secretion, as well as morphology with GTs.

Sequenced recretohalophyte genomes and their roles in understanding formation of secreting structures

Whole genome sequencing has been applied to different types of organisms including microorganisms, animals, and plants. It helps to explore the biological functions of genes, to unravel evolutionary history through comparative genome analysis and to advance breeding programs. Near 600 plant genomes have been sequenced and completely assembled (Kersey, 2019). High-quality plant genomes, with superior assembly and annotation, provide valuable insights that address issues pertaining to plant biology, by integrating transcriptomes from various perspectives. So far, 12 genomes of halophytes possessing secreting structures including epidermal bladder cells (EBCs), multicellular, bicellular and unicellular salt glands have been sequenced as shown in Table 3. Among these genomes, only five genomes have been assembled at the chromosome-level including M. crystallinum, A. marina, A. corniculatum, G. soja and L. bicolor. However, only the genomes of A. marina and A. corniculatum are available to the public.

Table 3. Genomes-sequenced of recretohalophytes with salt bladders or salt glands until 2023.

Species	Family	Class	Secreting structure	Assembly level	Available database	References
C. quinoa	Amaranthaceae-Chenopodiaceae	Dicot	EBCs	Scaffold	Index of /genomes/genbank/plant/Chenopodium_quinoa (nih.gov).. .. (NCBI)	(Jarvis et al., 2017; Zou et al., 2017)
M. crystallinum	Aizoaceae	Dicot	EBCs	Chromosome	Contact the authors of the reference	(Shen et al., 2022)
A. marina	Acanthaceae-Avicennioideae	Dicot	Salt glands (multicellular)	Chromosome	https://datadryad.org/stash/dataset/doi:10.5061/dryad.3j9kd51f5. .. .	(Friis et al., 2020; Natarajan et al., 2021)
A. corniculatum	Primulaceae-Myrsinaceae	Dicot	Salt glands (multicellular)	Chromosome	https://ftp.cngb.org/pub/CNSA/data3/CNP0001270/CNS0267886/CNA0017738/.. .. (CNGBdb)	(Feng et al., 2021; Ma et al., 2021)
G. uralensis	Fabaceae-Papilionoideae	Dicot	Salt glands (multicellular)	Scaffold	http://ngs-data-archive.psc.riken.jp/Gur-genome/download.pl.. .. (Dead link)	(Mochida et al., 2017)
G. soja	Fabaceae-Papilionoideae	Dicot	Salt glands or EBCs	Chromosome	http://www.wildsoydb.org/Gsoja_W05/; https://soybase.org/data/public/Glycine_soja/. ...	(Valliyodan et al., 2019; Xie et al., 2019)
L. bicolor	Plumbaginaceae	Dicot	Salt glands (multicellular)	Chromosome	Index of /genomes/genbank/plant/Limonium_bicolor (nih.gov).. .. (only assembly available from NCBI)	(Yuan et al., 2022)
A. littoralis	Poaceae-Chloridoideae	Monocot	Salt glands (bicellular)	Contig	https://doi.ipk-gatersleben.de/DOI/ac423f10-971e-481e-bcab-6ac261e27f5c/15d455e1-da91-4e78-82d8-7c7607cb05b9/2/1847940088.. ..	(Hashemi-Petroudi et al., 2022)
Z. matrella	Poaceae-Chloridoideae	Monocot	Salt glands (bicellular)	Scaffold	http://zoysia.kazusa.or.jp/.. ..	(Tanaka et al., 2016)
Z. japonica	Poaceae-Chloridoideae	Monocot	Salt glands (bicellular)	Scaffold		(Tanaka et al., 2016)
Z. pacifica	Poaceae-Chloridoideae	Monocot	Salt glands (bicellular)	Scaffold		(Tanaka et al., 2016)
O. coarctata	Poaceae-Oryzoideae	Monocot	Salt glands (unicellular)	Scaffold	http://ccbb.jnu.ac.in/ory-coar/.. ..	(Bansal et al., 2021)

Based on high-quality genomes, transcriptomes of EBCs of C. quinoa revealed the key genetic mechanisms in ion transport into EBCs. Ion transporters such as SLAH, NRT, ClC, NHX, and HKT1, as well as sugar transporters like SWEET, were found to be involved in the sequestration of ions and energy into specialized cells known as EBCs (Böhm et al., 2018). The EBC transcriptome of M. crystallinum comprised differentially expressed genes involved, among other things, in ion transport, energy metabolism and osmotic adjustment (Oh et al., 2015). Transcription factors of the bHLH family were identified in the high-quality genome of the mangrove Avicennia marina, followed by MYB, indicating these TFs play important roles in salt tolerance and are likely involved in salt gland development (Natarajan et al., 2021). L. bicolor genome contains trichome development-related transcription factors and some like the mentioned LbHLH and LbTTG1 have been characterized in more detail, providing new insights into salt gland development and salt tolerance (Yuan et al., 2022). These reports suggest that multi-omics, especially genome and transcriptome sequencing, plays a vital role in understanding the various aspects of secreting structures.

Perspectives

Until now, research progress on EBCs and salt glands has been slow, with many studies focusing on their physiological or morphological traits, while the mechanisms of their development and secretion remain poorly understood. Although EBCs can readily be isolated due to their larger size, and genomes of C. quinoa and M. crystallinum have been published, a suitable transformation system is still lacking. Despite the fact that the development of EBCs has been partially revealed through the use of mutants, a genetic transformation system of quinoa needs to be established. The role of EBC in salt tolerance also needs to be studied in more detail, given some controversial reports in the literature (e.g., Kiani-Pouya et al. 2017, 2019 vs Moog et al. 2022). It is plausible to suggest that the roles of EBC may be numerous, operating not only in damping salt load but also being a major storage reservoir for K⁺ and water.

Even though some genes involved in salt glands have been characterized, most of them are detected based on homology search, and their function was verified in a heterologous expression system, which may render misleading results. Applying multi-omics to mutants with salt gland abnormalities may be combined with genetic manipulation in the future to reveal mechanisms of salt gland formation and salt secretion. In light of the available genetic transformation system, recently published chromosome-level genome, and a wealth of previous studies, L. bicolor represents an excellent candidate for the study of salt gland development and secreting mechanisms, especially since it enables easy exploration of multicellular salt gland development and secreting mechanisms. Given that salt glands comprise different cellular types, the mechanisms regulating their development and secretion may differ to some extent, just as trichome development varies between Arabidopsis and rice. For a full understanding of salt gland evolution and their potential transfer to crops, it is necessary to study the development and secreting mechanisms of salt glands with different cellular types. O. coarctata, a wild relative of rice, is a type of unicellular salt gland and only secreted on the adaxial leaf surface, which makes it a suitable model for studying how salt glands form and how salt is secreted through them using conventional and modern methods. The Aeluropus family can be regarded as a representative of the bicellular salt glands that can be easily and quickly reproduced using cottage. G. soja, as a wild relative of cultivated soybean G. max, has the potential of making it possible to apply salt bladders or glands to G. max based on multiple-omics as there is a high homology between them, making it easy to exchange genetic materials (Li et al., 2014; Singh, 2019; Xie et al., 2019). Additionally, cultivated soybean genetic transformation system and CRISPR/Cas9 can be further applied to wild soybeans, to understand mechanisms underlying salt secretion. However, the primary challenge in studying salt gland development and secreting mechanisms is the difficulty of isolating salt glands from multiple epidermal cells. Salt glands do exhibit autofluorescence under ultraviolet excitation at the wavelength of 330-380 nm (Deng et al., 2015), which can be utilized in combination with laser capture microdissection (LCM) to acquire salt glands in the future. Following the acquisition of salt glands, multi-omics analysis can be employed to identify key genes responsible for salt gland development and salt secretion, which can subsequently be confirmed through transformation systems.

Additional information

Funding

This work was supported by Joint Research Projects between Pakistan Science Foundation and National Natural Science Foundation of China [Grant No.: 31961143001].