Environmental stresses influence plant metabolism and physiological activities through osmotic balance, ion homeostasis, signal transduction, redox balance, gene expression and enzyme activity modifications etc [34, 35]. Usually, generation of ROS species and toxicity mechanisms are similar in glycophytes and halophytes; however, the strategies for detoxification and prevention differ in terms of total antioxidant activity and isoenzymic-form expressed in response to salinity. Plants respond to oxidative stress primarily through an endogenous defensive mechanism comprised of various enzymatic and non-enzymatic antioxidants. [36, 37]. A steady-state balance between the antioxidant scavenging system and accumulation of ROS in plant cells, enables the optimal ROS level in the cell for adequate redox biology and normal plant growth regulation processes. In our studies, salt stress induced oxidation stress was scavenged by enhanced activities of these antioxidant enzymes as well as the antioxidants accumulation in both the halophytic plants where SOD activity was more in highly salt tolerant, U. setulosa than D. annulatum. While comparing both the halophytes, the free radicals generated due to salinity are initially converted to H2O2 by SOD as we can see from the accumulated H2O2 content and higher expression of SOD. In D. annulatum, this H2O2 is further reduced by catalase while in U. setulosa higher peroxidase activity correlates with H2O2 content which further is carried forward by AsA-GSH pathway. Ascorbate and glutathione contents were 2–3 fold higher in U. setulosa than D. annulatum depicting more detoxification of H2O2 at higher salinity levels. Hence, we can briefly summarize that at lower levels of salinity SOD and catalase detoxify the free radicals in D. annulatum while at higher salinity levels, it is being dismutated through SOD and AsA-GSH pathway in U. setulosa and hence, the later is highly salt tolerant.
During ROS scavenging, The enzyme activities of APX, MDHAR, DHAR, and GR define the AsA-GSH pathway along with the concentration of antioxidant metabolites, ascorbate and glutathione. [38]. Ascorbate peroxidase enzyme breaks down reactive H2O2 into water with release of MDHA, which is quickly dissociated into ascorbate and DHA. Further, glutathione (GSH) catalyzes reduction of Dehydroascorbate (DHA) into ascorbate yielding oxidized glutathione GSSG. This GSSG is finally reduced by glutathione reductase (GR). The reduced of AsA and GSH antioxidants, plays important role in conferring stress tolerance to plants. [38]. Here, we can see that the expression level alongwith enzyme activity of SOD, POX, Asc Peroxidase, DHAR, MDHAR and GR are prominent in U. setulosa at higher salt levels of EC 40 dSm− 1 and 50 dSm− 1 while the Asc-GR pathway seems to be less active in D. annulatum at lower levels of salinity. Superoxide dismutase (SOD) plays an important role to define the specific H2O2 ‘signature’ in halophytes and hence, its higher intrinsic activity triggers a cascade of physiological and molecular responses for further stress signaling, thereafter, other antioxidant enzymes detoxify this H2O2 [39].
The first effect of ROS accumulation under abiotic stress exacerbate membrane damage through lipid peroxidation which is a non-enzymatic autoxidation processes. Here, we measured lipid peroxidation in form of malondialdehyde (MDA) content which accumulated more in D. annulatum (Table 2) than U. setulosa, the first being more sensitive to salinity whereas, H2O2 content was higher at high salt levels in U. setulosa than in D. annulatum (Table 2). Superoxide dismutase acts as the first defensive barrier against oxidative stress by dismutating superoxide radicals into less toxic H2O2 [40]. In our studies also, we have seen that SOD activity increases with increasing levels of H2O2 in both the plants with higher activities in tolerant halophyte U. setulosa than the moderately tolerant D. annulatum. Catalase defends cells against H2O2 by catalyzing it into H2O and O2. Higher catalase activity is correlated with enhanced gene expression reducing the oxidative damage, owing to this enzyme's protective function. In present experiment, the activity of catalase is more than peroxidase in D. annulatum than U. setulosa, probably, H2O2 is being detoxified more through catalase (Fig. 1). In addition to its scavenging role in detoxification of H2O2, peroxidase is also involved in lignification and cell-wall biosynthesis. From the differential expression of DEGs under oxidative stress, we can see peroxidase activity due to oxidative stress involved in hydrogen peroxide catabolic process as the integral component of membrane (Supplementary table 1) and extracellular region including L-ascorbate peroxidase, glutathione peroxidase and catalase activity. In DEG analysis, we found that the transcripts upregulated for peroxidase in U. setulosa are mainly involved in biosynthesis of phenylpropanoids which are involved in most of the plant responses against biotic and abiotic stresses. Polymers based on phenylpropanoids, such as lignin, tannins or suberins impart mechanical and environmental resistance in angiosperms and gymnosperms against wounding or drought or other stress. In the present study, the upregulation of these transcripts at high salinity also indicate more membrane stability which again proves the tolerance potential of these halophytes. For cell redox category, protein disulfide oxidoreductase activity along with isomerase activity was upregulated (Supplementary table 3). The families of thioredoxin, protein disulfide-isomerase (PDI), glutaredoxin, DsbA (disulfide-bond forming) and their homologs constitute the protein disulfide oxidoreductases. As per its functional annotation, this protein has isomerase activity too in addition to oxidative and reductive activities. In mungbean also, increased levels of superoxide radicals, H2O2 content along with higher activities of enzymes; SOD, CAT, APX, GR have been reported with increased electrolyte leakage and lipid peroxidation due to NaCl [37]. In other plants also like, Brassica juncea [41], tomato [42] and Triticum aestivum L [43] higher activities of antioxidant enzymes have been reported. The changes in these enzymes are related with plant responses to salt induced iso-osmotic stress via balancing the cellular redox.
H2O2 accumulation is extremely toxic to the cell because it is capable of passing through biological membranes and infiltrating other subcellular compartments. As a result, instant detoxification of H2O2 through ascorbate peroxidase is critical for a robust ROS system that could also regulate the cellular levels of H2O2 in green leaves. In our study also, significantly higher ascorbate peroxidase activity in both the halophytes indicates the scavenging of H2O2 through APX pathway. The enhanced SOD activity alongwith the other major antioxidative enzymes such as GR, MDHAR and DHAR reflects the involvement of these antioxidative enzymes in detoxifying effect of ROS and hence, governing salt tolerance of Urochondra and Dichanthium at different levels of salinity.
A positive effect of both components, enzymatic and non-enzymatic, in imparting salt tolerance has been explored earlier in various plants including halophytes. For example, significant increases in activities of CAT and APX have been observed in sapodilla rootstock (Manilkara zapota (L.) P. Royen) with exposure of rootstocks to diluted seawater of EC 12 dSm− 2 [44]. In salt tolerant and sensitive rice varieties, increasing transcript levels of Mn-SOD and GR genes (P ≤ 0.05) are positively correlated with NaCl concentrations. At 30 and 90 mM NaCl, APX gene expression levels increased in Mevlu¨tbey rice variety which decreased at higher NaCl levels of 150 and 210 mM [45]. The previous reports on other halophytes such as Suaeda fruticosa [46], Limonium delicatulum [47], Sporobolus marginatus [40], Haloxylon salicornicum [48], also revealed similar report for the enhanced enzyme activity under salinity conditions which showed the stronger capability of these halophytes to remove ROS by protecting from oxidative damage and maintained redox homeostasis. ROS activation was linked to varying tolerance in the genus Juncus towards salinity and drought in three species viz. J. maritimus, J. acutus (both halophytes) and J. articulatus (salt sensitive) [1]. Oxidative stress was created in all these plants due to water and salt stress but higher malondialdehyde accumulation was reported in J. articulates and whereas the other two halophytes J. maritimus and J. acutus showed better tolerance in terms of efficient ROS system with increased activities of superoxide dismutase and glutathione reductase. Inter & intra species specific variations were observed for ascorbate peroxidase activity depending on treatment. To overcome the oxidative stress, redox homeostasis was maintained in Suaeda corniculata through highly active APX dependent ascorbate-glutathione and peroxiredoxin pathways [49]. The genes of antioxidant system for SOD, APX, GR, DHAR, MDHAR from Pennisetum glaucoma (Pg) were transferred in tomato seedlings and a significantly higher activities of these enzymes was observed in the transgenic plants exposed to drought and salinity respectively. Additionally, membrane stability in terms of reduced electrolytic leakage and lower malondialdehyde contents were observed [34].
Generally, ROS in plants regulate signal transduction cascade in response to abiotic stress through downstream signaling of redox-sensitive transcription factors and receptor proteins with direct inhibition of phosphatase [50]. This signaling starts with calcium and phospholipid with further network of transcription factors, serine/threonine protein kinase, NADPH oxidase and MAPK cascades [51] as has been reported in other halophytes including Sporobolus virginicus [52], Mesembryanthemum crystallinum [53], Halogeton glomeratus [54] and Suaeda fruticose [55] through comparative transcriptomics under salt stress. Through such studies, several transcription factors including WRKY, DREB, NAC and basic leucine zipper (bZIP) were evaluated for imparting salt tolerance [56]. In our studies, a significant enrichment of various components of ROS system were revealed [Supplementary tables 1–4]. In total, 66 and 276 differentially expressed genes (DEGs) for ROS scavenging and signaling were discovered at higher salt concentrations in the halophytes, D. annulatum and U. setulosa respectively. In response to stress, the proteins coding for these transcripts include heat shock proteins, universal stress proteins, calmodulin binding proteins, Dehydrin/LEA group-2 like proteins, putative trehalose-6-phosphate synthase and Abscisic acid stress ripening proteins etc. in U. setulosa and in D. annulatum mainly coding for calmodulin binding proteins. We identified few genes for cell redox homeostasis in these two halophytes mainly coding for glutaredoxin family proteins, disulfide isomerase, protein disulfide isomerase-like, putative nucleoredoxin, Thioredoxin-like-3-1-chloroplastic with molecular function of cell redox homeostasis. During stress conditions, genes that regulate redox reactions are mostly involved in protecting the cell behavior and environment. Similar to our findings, the differential expression analysis revealed an increase in glutathione-S-transferase tau1 and glutathione transferase in Suaeda fruticosa [55], Salicornia europaea [57], Suaeda maritime, Reaumuria trigyna [58]. GST gene from Suaeda salsa was transformed in Arabidopsis which enhanced the salt tolerance capacity in transgenics. Increased Glutathione content has also been reported in Arabidopsis under salt stress contributing in increasing salt tolerance [59]. In our findings also, most of the genes coding ROS system were expressed upon exposure to salinity involved in various protecting and redox pathways. Cumulatively, we can summarize that at different salt levels, U. setulosa tend to maintain membrane integrity and steady levels of ROS with prominent role of SOD and Asc-GSH pathway than moderately salt tolerant halophyte, D. annulatum. U. setulosa is able to tolerate higher salt concentrations than other halophytes, hence, it can be further explored for gene mining for salt tolerance since the model halophyte, Thellungiella halophile, salt cress has been evaluated at lower levels of saline treatments for short durations.