The Halophyte Seashore Paspalum Uses Adaxial Leaf Papillae for Sodium Sequestration

Salinity is a growing issue worldwide, with nearly 30% of arable land predicted to be lost due to soil salinity in the next 30 years. Many grass crops that are vital to sustain the world’s caloric intake are salt sensitive. Studying mechanisms of salt tolerance in halophytic grasses, plants that thrive in salt conditions, may be an effective approach to ultimately improve salt-sensitive grass crops. Seashore paspalum (Paspalum vaginatum) is a halophytic Panicoid grass able to grow in salt concentrations near that of seawater. Despite its widespread cultivation as a sustainable turfgrass, the mechanism underlying its ability to retain high Na⁺ concentrations in photosynthetic tissue while maintaining growth remains unknown. We examined the leaf structure and ion content in P. vaginatum ‘HI10’, which shows increased growth under saline conditions, and Paspalum distichum ‘Spence’, which shows reduced growth under salt, to better understand the superior salt tolerance of cv HI10. A striking difference between cv HI10 and cv Spence was the high steady-state level of K⁺ in cv HI10. Imaging further showed that the adaxial surface of both cv HI10 and cv Spence contained dense costal ridges of papillae. However, these unicellular extensions of the epidermis were significantly larger in cv HI10 than in cv Spence. The cv HI10 papillae were shown to act as Na⁺ sinks when plants were grown under saline conditions. We provide evidence that leaf papillae function as specialized structures for Na⁺ sequestration in P. vaginatum, illustrating a possible path for biotechnological improvement of salt-sensitive Panicoid crops with analogous leaf structures.

About 20% of irrigated land is considered saline, with the amount of saline soils increasing worldwide (Mayak et al., 2004). This is due to increased irrigation in agricultural fields necessitated by more frequent droughts due to climate change. This trend is alarming due to the high salt sensitivity of most crop species that we rely on for vital resources. Yield reduction in crops in saline soils amounts to losses on the order of 12 to 27.3 billion U.S. dollars annually (Qadir et al., 2014). Thus, the improvement of salt tolerance in plants will become key in the coming decades. Breeding salt-tolerant crops is a cost-effective approach to improve growth in saline soils. Although much work has focused on breeding salt-tolerant species, progress in this area has been slow due to the complex genetic and physiological nature of the salt response. Furthermore, most research has been conducted on glycophytic model systems that are salt sensitive (Munns and Gilliham, 2015). Unraveling the salt-tolerance mechanisms in halophytes, species that can complete their life cycle in 200 mm salt concentrations, and transferring these pathways into glycophytes is therefore of great interest (Rajalakshmi and Parida, 2012; Roy and Chakraborty, 2014).Both glycophytes and halophytes have evolved a multitude of salt-tolerance mechanisms, including sodium (Na⁺) exclusion, sequestration, and secretion; osmolyte production; ion homeostasis; and reactive oxygen species (ROS) detoxification (Meng et al., 2018). Often, mechanisms present in glycophytes, such as osmolyte production and Na⁺ exclusion, are utilized in halophytes at higher efficiency (Wyn Jones and Storey, 1981; Grieve and Maas, 1984). However, halophytes also use mechanisms that are absent in glycophytes. Salt sequestration and secretion via salt glands is a halophyte-specific mechanism of coping with salt (Flowers and Colmer, 2008). Salt glands are found in over 50 species in 14 angiosperm families with four subtypes: epidermal bladder cells, complex multicellular glands, bicellular glands, and unicellular glands (Dassanayake and Larkin, 2017). The Poales order contains ∼8% of all halophytes (Flowers et al., 2010) and has therefore been the focus of much salt-gland-focused work (Ceccoli et al., 2015). As salt tolerance has independently evolved >70 times in grass lineages (Bennett et al., 2013), studying these salt sequestering/secreting structures in grasses is an excellent approach to better understand salt tolerance mechanisms in halophytes.Most structural and physiological work on salt glands in grasses has been conducted in the Chloridoideae and Oryzoideae subfamilies. Grasses carry either unicellular or bicellular glands, often referred to as glandular trichomes or microhairs, on the leaf surface (Dassanayake and Larkin, 2017). Microhairs have been observed on the leaf surface in all grass subfamilies except the Pooideae, and have evolved diverse functions including the sequestration or secretion of substances such as callose and heavy metals (Burke et al., 2000; Ceccoli et al., 2015). Unicellular structures on the adaxial leaf side able to secrete salt are only found in the Oryzoideae wild rice species Porteresia coarctata (Flowers et al., 1990; Sengupta and Majumder, 2009). Salt glands in the Chloridoideae are bicellular, consisting of a cap cell and a lower basal cell, both of which are dense in cytoplasm and mitochondria (Ceccoli et al., 2015). The cuticle is thickened above the cap cell in some species, forming a cuticular chamber used for storing secreted salts (Amarasinghe and Watson, 1988). In the Panicoideae, a few cases of Na⁺ secretion have been reported (McWhorter et al., 1995; Ramadan and Flowers, 2004), but to date, no sequestration structures have been identified.The Panicoideae subfamily includes the agronomically important food crops maize (Zea mays) and sorghum (Sorghum bicolor) in addition to the biofuel grasses miscanthus (Miscanthus sinensis), switchgrass (Panicum virgatum), and sugarcane (Saccharum officinarum). One of the most salt-tolerant species in the Panicoideae is the halophyte seashore paspalum (Paspalum vaginatum). It is cultivated as a turfgrass worldwide and derives its popularity from its ability to be irrigated with brackish water. P. vaginatum can survive in salt concentrations near that of seawater (Uddin et al., 2012) and uses osmolyte production, ion homeostasis, and Na⁺ exclusion to cope with salt stress (Peacock and Dudeck, 1985; Lee et al., 2008; Guo et al., 2016). However, its ability to maintain growth while accumulating high levels of Na⁺ in leaf tissue remains perplexing.Here, we studied the leaf structure and Na⁺ sequestration in ‘HI10’, a P. vaginatum cultivar, and ‘Spence’, a Paspalum distichum cultivar. P. vaginatum and P. distichum are closely related (and possibly the same species; Eudy et al., 2017), and constitute group “Disticha” in the tribe Paspaleae. P. distichum is less salt tolerant than P. vaginatum and is typically found in freshwater habitats (Leithead et al., 1971). P. vaginatum and P. distichum therefore represent a useful species pair to study salt tolerance. Furthermore, their salt responses can be compared with those of sorghum, a Panicoid glycophyte. Our main research objective was to identify the phenotypic and physiological factors that contribute to the differential tolerance to salt stress of the two Paspalum spp. cultivars and sorghum ‘BTx623’. We show that both Paspalum species contain dense rows of translucent papillae on the adaxial surface. The papillae are unicellular protrusions from epidermal cells and are much larger in cv HI10 than in cv Spence. We further demonstrate that the papillae sequester Na⁺ under salt stress. This study thus provides evidence of Na⁺ sequestration in specialized leaf-borne organs within the Panicoideae.