Salt stress is a major abiotic threat to plants and has severe effects on agricultural productivity worldwide . Salinity induces ion imbalance, hyperosmotic stress and oxidative damage in plants . Plants have developed complex adaptive mechanisms to cope with the salt stress, such as photosynthetic adjustments, synthesis of osmolytes (e.g., glycine betaine, soluble sugar and proline), and ion homeostasis . In the past years, the salinity-responsive mechanisms in leaves and roots from a number of plant species have been investigated using molecular genetics and different omics strategies [4–9]. In plants, the salt signal perception and transduction, detoxification of reactive oxygen species (ROS), ion uptake/exclusion and compartmentalization, salt-responsive gene expression, protein translation and turnover, cytoskeleton dynamics, cell wall modulation, as well as carbohydrate and energy supply have been investigated in various organs [5, 6]. However, these differentiated organs (e.g., leaves and roots) contain heterogeneous cell types and developmental stages, which may exhibit contrasting sensitivity to salinity. Therefore, it is difficult to determine the cell specific characteristics of salt tolerance when using leaves and roots as materials .
Cultured cells are a good model system for investigating cell-specific metabolism because they can be synchronized. Callus obtained by in vitro culture is a group of unorganized cell mass, which has capability to regenerate into a whole plant through somatic embryogenesis and organogenesis. Importantly, callus is an excellent material for genetic transformation in molecular genetics studies. Physiological alterations in calli obtained from sugarcane (Saccharum officinarum) [11–13], wheat (Triticum durum) , rice (Oryza sativa) [14, 15], and cotton (Gossypium hirsutum)  under salinity, osmosis or oxidant conditions were investigated to reveal the stress-responsive mechanisms at cell levels. When being exposed to NaCl stress, sugarcane callus reduced its growth and cell viability, although the cells have the ability to accumulate proline and glycine betaine, and secrete Na+ . The growth of sugarcane callus was also decreased under mannitol-induced osmotic stress, likely due to the decreased K+ and Ca2+ concentrations . The salt-tolerant callus selected from sugarcane cultivar CP65–357 can accumulate more K+, proline and soluble sugar, which could facilitate ion and osmotic homeostasis . In general, proline accumulation is an important strategy for osmotic adjustment. However, it has been regarded as an injury symptom rather than an indicator of tolerance in rice callus under salt stress . Among calli from durum wheat (T. durum) cultivars with different salt-tolerance capabilities, salt-altered relative growth rate (RGR) and cell viability were correlated, and an induced non-phosphorylating alternative pathway played an important role in salt tolerance . The calli from salt-tolerant wheat cultivar were able to recover after stress relief, and ATP-production was crucial for its growth maintenance . Also, in the callus from NaCl-tolerant cotton, the activities of antioxidant enzymes (e.g., ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR)) were induced, and ROS and nitric oxide played important signaling roles in the course of establishing NaCl tolerance . However, the sophisticated salinity-responsive signaling and metabolic pathways in callus are still unclear.
High-throughput proteomics is a powerful platform for revealing the protein abundance patterns during plant development and environmental responses . Two dimensional electrophoresis (2DE) gel-based and isobaric tags for relative and absolute quantification (iTRAQ)/tandem mass tag (TMT)-based quantitative approaches have been applied to reveal molecular changes during callus development, differentiation and somatic embryogenesis of different plant species, such as sugar cane (Saccharum spp.) [18, 19], maize (Zea mays) [20–22], rice (Oryza sativa) , oil palm (Elaeis oleifera × Elaeis guineensis) , Valencia sweet orange (Citrus sinensis) , Cyclamen persicum , Vanilla planifolia [27, 28], and lotus (Nelumbo nucifera Gaertn. spp. baijianlian) . These studies have improved understanding of the molecular regulatory roles of H+-pumps (i.e., P H+-ATPase, V H+-ATPase, and H+-PPase), sucrose and pyruvate accumulations, ROS homeostasis, protein ubiquitination, phytohormone and growth regulators (e.g., auxin, cytokinin, abscisic acid and polyamine putrescine) in embryogenic competence acquisition in callus. Importantly, some critical proteins identified in these studies are potential biomarkers for embryogenic competence acquisition, and their functions need to be further investigated . To date, proteomic studies of callus salt tolerance have rarely been reported.
Alkaligrass (Puccinellia tenuifora) is a monocotyledonous halophyte with high salinity, alkali and chilling tolerance. It can grow under 600 mM NaCl and 150 mM Na2CO3 (pH 11.0) for six days , and can survive chilling stress . Our previous proteomics and physiological studies have reported the salt-/alkali-responsive mechanisms in leaves and roots in response to NaCl (50 mM and 150 mM for seven days) , Na2CO3 (38 mM and 95 mM for seven days; 150 mM and 200 mM for 12h and 24h) [33–35], and NaHCO3 (150 mM, 400 mM and 800 mM for seven days)  stresses. We found alkaligrass accumulated Na+, K+ and organic acids in vacuoles, as well as proline, betaine and soluble sugar in the protoplasm to maintain osmotic and pH homeostasis in response to salt stress [32, 37]. In these differentiated organs, the fine-tuned mechanisms of signal transduction, ion and osmotic homeostasis, ROS scavenging, transcription and protein synthesis, as well as energy and secondary metabolisms were quite different. However, the salinity-responsive mechanisms in the unorganized callus of alkaligrass have not been reported.
In this study, we investigated the physiological and proteomic characteristics of alkaligrass callus in response to NaCl treatments. The molecular modulations of ROS scavenging, osmotic homeostasis, energy supply, as well as gene expression and protein processing were active in callus under salinity stress. Our results provide new insight into the NaCl response in undifferentiated plant cells, and may have potential applications in the engineering and breeding of salt-tolerant plants.