Salinity signal transduction and cytoskeleton dynamics in callus
Callus development is sensitive to salinity, and many signaling and metabolic pathways were modulated by the salt treatments. In alkaligrass callus, the signal transduction and cytoskeleton dynamics were altered due to several NaCl-regulated proteoforms of 14-3-3 protein and actin (Fig. 7a). 14-3-3 proteins are multifunctional proteins that regulate diverse downstream target proteins in signal transduction, vesicle trafficking, ion transport, chromatin modulation, and various metabolic pathways in plant response and adaptation to stress . The abundances of two 14-3-3-like proteins and TaWIN1 protein were decreased in salt-stressed callus and leaves of alkaligrass . 14-3-3 GF14λ protein was shown to interact with somatic embryogenesis receptor kinase 1 (SERK1) in signal transduction cascade regulating Arabidopsis embryogenic development , and to function in the regulation of actin cytoskeleton, cell wall remodeling, and the cell cycle during early stages of somatic embryogenesis of oil palm . Importantly, 14-3-3 proteins were found to trigger programmed cell death in callus from grape , barley  and tomato  in response to biotic stresses, such as infections of A. tumefaciens and powdery mildew. In salt stress, whether the decrease of several 14-3-3 proteins would help to alleviate salt sensitivity in callus is not known. It needs to figure out the downstream interacting proteins for understanding the 14-3-3 protein- regulated signaling pathways during callus salt response.
Cytoskeletal dynamics were revealed from salt-altered abundance patterns of three actin species (two salt-induced and one salt-reduced). Actin is necessary for callus formation in Arabidopsis  and different oil palm species [24, 42]. The depolymerization of the actin cytoskeleton was taken as an important strategy for stress adaptation, triggering the execution of programmed cell death in non-embryogenic callus from maize [21, 43]. It has been reported that Arabidopsis TCP1 assisted the actin and tubulin protein folding as molecular chaperone to keep the stem cell maintenance , which implied that the salinity-induced TCP1 in alkaligrass callus would facilitate the cytoskeletal dynamics to cope with salt stress.
Salinity-responsive ROS scavenging pathways in callus
Salt-induced ROS are capable of causing cellular damage by disrupting membrane integrity, degradation of proteins, and inactivation of enzymes . In addition, certain levels of ROS can function to signal cell dedifferentiation and promote somatic embryogenesis in embryogenic callus (EC) . ROS homeostasis in plant callus is fine-tuned . For example, in grape (Vitis vinifera) callus, strong APX pathway was triggered in embryogenic callus, whereas CAT pathway was utilized in non-EC . Our study revealed the ROS signal and scavenging networks were sophisticatedly tuned in alkaligrass callus.
The increase of SOD activity contributes to dismutase intracellular O2•- to H2O2 in alkaligrass callus under NaCl stress (Fig. 3b). Similarly, it has been shown that callus from a NaCl-tolerant cotton cultivar exhibited significantly salt-enhanced SOD activity, whereas callus tissue from a NaCl-sensitive cultivar exposed to salt stress showed little change in the SOD activity . Besides, high SOD accumulation was detected in EC compared to non-EC from grapevine (Vitis vinifera L. cv. Cabernet Sauvignon) . This result was quite different from the NaCl-/ Na2CO3- decreased SOD activities in alkaligrass leaves and roots [30-32]. This indicated that the undifferentiated salt-tolerant callus from halophyte alkaligrass initially and specifically employed SOD pathway for ROS homeostasis, when comparing with the salt-sensitive callus and these differentiated organs.
To further maintain homeostasis, the increased H2O2 is scavenged in diverse pathways. CAT pathway can directly dismutase H2O2 to H2O and O2 in peroxisomes. In salinity-/alkali-stressed alkaligrass, CAT activities were reduced in callus and short-term (12h and 24h) salt-treated leaves and roots [32, 33], but induced in long-term (7 days) salt-treated leaves [30, 31]. POD also catalyzes the reduction of H2O2 using various substrates, such as phenolic compounds, lignin precursors, auxin and secondary metabolites. The NaCl-induced POD activity in alkaligrass callus implies that it is crucial for the protection of callus cells. Similarly, a wide variety of peroxidases were also induced in sugarcane and maize embryogenic callus [19, 22], and peroxidases were more active in ECs from palm (Phoenix dactylifera), lettuce (Lactuca sativa) and Medicago truncatula [48-50]. In addition, the AsA-GSH cycle catalyzed by APX, MDHAR, DHAR and GR participates in the removal of H2O2, which was implicated in the maintenance of cell wall plasticity and the stimulation of organized cell division . In alkaligrass callus, the activities of APX, MDHAR and GR were NaCl-induced, but DHAR activity was NaCl-reduced. This result was similar to that in leaves under Na2CO3-treatmenf for 12h and 24h . In addition, the amount of APX and an isoform of MDHAR, as well as the AsA content were all increased in the alkaligrass callus after salt stress. These data suggest that the induced AsA-GSH cycle was critical in regulating the ROS levels in response to salinity stress, and also in maintaining the ability for stimulating cell differentiation upon somatic embryo formation .
The increase of GSH and decrease of GSSG are consistent with the induced GPX activity and decreased GST activity in alkaligrass callus and similar to the changes observed in short-term Na2CO3-treated roots . The salt-induced cysteine synthase in alkaligrass callus would facilitate the biosynthesis of cysteine which serves as a precursor for the synthesis of various sulfur-containing metabolites including GSH [25, 53]. It has been reported that cysteine synthase was highly expressed in EC to promote dedifferentiation . Another salinity-increased protein ferritin plays an important role in iron sequestration, preventing the reaction of iron with ROS to cause severe oxidative stress . Clearly, among the complicated ROS pathways in alkaligrass callus, NaCl-induced AsA-GSH cycle and POD pathway function to enhance oxidative stress tolerance and maintain cell dedifferentiation, and thereby promote somatic embryo formation .
Diverse osmotic regulation strategies in salt-stressed callus
Under salinity, cell membrane integrity and membrane lipid composition were altered to modulate signal transduction, osmotic homeostasis and cell structure. MDA contents in cells are often used to evaluate cell membrane damage. In alkaligrass under 50 mM and 150 mM NaCl treatments, MDA content was increased in callus (Fig. 1i) but not significantly altered in leaves . However, MDA contents were increased in leaves under alkali (Na2CO3 and NaHCO3) stress [31, 34]. This indicates different tissues may respond differently to neutral salt stress and alkali stress.
To cope with osmotic imbalance, diverse compatible osmolytes accumulated in callus. In salt-stressed alkali callus, the contents of proline, soluble sugar, and glycine betaine were significantly increased (Fig. 2a-c), and BADH involved in glycine betaine biosynthesis was increased in abundance (Additional file 2). Proline is considered to be a compatible osmoticum, and also serves as a storage sink for carbon and nitrogen, stabilizes subcellular structures and buffers cellular redox potential for protecting from oxidative damage under stress [55-57]. In leaves of alkaligrass, the proline content was induced by NaCl  and NaHCO3 . Moreover, proline content was also increased in calli form rice (Oryza sativa cv. KDML 105)  and two cultivars of Medicago sativa (cv. Yazdi and cv. hamedani)  under NaCl stress. In sugarcane (Saccharum sp.), proline content is higher in salt-tolerant callus obtained by in vitro selection than in non-selected callus under NaCl stress . Similarly, a salt-adapted tobacco callus accumulated more proline than unadapted callus after salt shock . However, in the callus from three sugarcane cultivars with different salt tolerance ability, the salt-resistant cv. R570 accumulated less proline than intermediary salt-resistant cv. NCo310 and salt-sensitive cv. CP59-73 . This perhaps indicates that proline accumulation was merely a stress response, rather than a stress tolerance trait. Similar phenomenon was also reported in callus from tomato , rice , and Fraxinus angustifolia  under NaCl stress. All these suggest that the contribution of proline to osmotic adjustment exhibits different significance in callus from diverse plant species and under different stress conditions. Sometimes, soluble sugars and glycine betaine may play more important roles. For example, soluble sugar content was induced in alkaligrass callus and leaves under NaCl or Na2CO3 stress. Besides, the salt-tolerant callus of sugarcane accumulated more soluble sugars under NaCl stress . However, the soluble sugars did not accumulate in callus from salt-resistant wheat cv. belikh . In NaCl-stressed alkaligrass callus, the glycine betaine content was significantly increased, which was consistent with the result from NaCl-stressed sugarcane callus  and the salinity- or alkali (e.g., Na2CO3, and NaHCO3)- stressed alkaligrass leaves [30, 33, 34].
Active glycolysis for energy supply in callus
Callus tissues grow fast and have high cell division rate. A large amount of energy is needed via glycolysis, the TCA cycle, and subsequent energy via the mitochondrial respiratory chain . In the EC of C. persicum and V. planifolia, the glycolytic enzymes were significantly increased due to energy demands of rapid growth and cell division [27, 63]. In this study, eight enzymes involved in glycolysis, PPP, TCA cycle, and other sugar metabolism pathways were affected in alkaligrass callus in response to NaCl stress (Fig. 5d and 7d, Additional file 2). Among them, the NaCl-induced proteoforms of GAPDH, ENO and PDC suggest that glycolysis was enhanced . Similarly, the protein abundance of GAPDH was increased during Vanilla planifolia callus development [27, 28], and the gene expression level of GAPDH was also induced significantly in grape EC . Besides, ENOs were induced in EC relative to non-EC of Vitis vinifera , as well as in the developing embryos of spruce (Picea asperata) under oxidative stress . All these imply that the increase of glycolysis was inclined to enhance the cellular division and differentiation during callus development and somatic embryogenesis processes . However, the NaCl-decreased IDH and MDH indicate that the TCA cycle was salinity-inhibited in alkaligrass callus. Consistently, the abundance of various enzymes involved in the generation of acetyl-CoA for the subsequent TCA cycle were lower in EC than in non-EC of maize . In addition, NaCl reduced several mitochondria-localized ATP synthase α and β in alkaligrass callus, which were also decreased in EC when compared in non-EC from maize and grape [21, 38]. This implies that the energy supply mainly rely on glycolysis but not TCA cycle, because the demand for energy is lower in slow growth of EC under salt stress.
Importantly, the SUS is a key reversible link in sucrose metabolism between respiration, carbohydrate biosynthesis, and carbohydrate utilization . In this study, two NaCl-induced SUS could facilitate sucrose flux for energy supply and osmotic homeostasis in alkaligrass callus. In addition, NaCl-altered alpha-galactosidase (α-Gal) may also help sugar flux for the cell wall expansion in callus to cope with salinity (Fig. 5d and 7d, Additional file 2).
Protein synthesis and processing are necessary for callus
Protein synthesis and processing are active during rapid cell reprogramming of somatic embryogenic growth [68, 69]. The proteins involved in protein synthesis and processing accounted for 20% of total proteins in EC and non-EC from saffron (Crocus sativus) . The changes to protein profiles observed in our proteomics results imply that transcription, translation, and protein processing in alkaligrass callus were all perturbed by salinity. The altered transcription can be reflected by the NaCl-induced DNA repair protein RAD23, as well as NaCl-reduced Macro domain-containing protein VPA0103 and RH2 [64, 70]. In addition, NaCl-increased EF2 functions in the GTP-dependent ribosomal translocation step during translation elongation. Our PPI analysis showed that EF2 may link with a number of proteins in energy supply, cytoskeleton, protein folding, and ROS scavenging (Fig. 6). This implies that the enhanced protein synthesis may facilitate diverse pathways in alkaligrass callus to cope with salinity.
In embryogenic development and callus stress response, removal and refolding of unnecessary and misfolded polypeptides are vital for cell reprogramming . In alkaligrass callus, we found that salt increased HSP 90 and TCP1, as well as salt-decreased HSP70 and Hsp70-Hsp90 organizing protein 1 (HOP). Importantly, our PPI prediction implies that these salt-responsive molecular chaperonins interact with most proteins in charge of energy metabolism, protein synthesis, and ROS scavenging in callus (Fig. 6). The accumulation of HSP70 and other HSPs was also found in EC when compared with non-EC from grape [38, 71]. Interestingly, different members of HSP family exhibited diverse abundance patterns during embryo development. For example, HSP60 and HSP101 were typical for the early somatic embryos, while HSP20 and HSP70 marked the late stage of embryogenesis . The assorted chaperon-depended modulations of peptide structure indicate that the assembly/structure of the newly-synthesized peptides and stabilization of the mature proteins were dynamically regulated during in callus development and stress response [28, 71, 72].