Molecular response of canola to salt stress: insights on tolerance mechanisms

Survey methodologys

We searched literature relevant to the topic of the article using Google Scholar and PubMed. Key words such as “canola,” “Brassica,” “salt stress,” “salinity,” “tolerance mechanism,” “proteomic analysis,” “transcriptomic analysis,” plant salt-tolerance mechanism,” “gene regulation,” “proteome profile,” “signal transduction,” and “gene regulatory mechanism” were used to search. The combination of these key words was also used. Then, the articles were screened and used as references for the review.

Signal transduction

In canola roots, it is proposed that Ras-related small GTP-binding proteins mediate salt stress signaling. This protein has been identified by proteomic analysis of canola cultivars under salt stress (Banaei-Asl et al., 2015). The Ras-related small GTP-binding protein has also been identified in canola leaves under salt stress (Banaei-Asl et al., 2016; Bandehagh et al., 2011). It has previously been indicated that this protein acts as a signaling molecule, responding to salt stress, and is associated with other proteins such as G-protein-couples receptors (GPCRs) (Vernoud et al., 2003). The identification of Ras-related small GTP-binding protein which is upregulated in response to salt stress in canola (Banaei-Asl et al., 2015) may, in turn, imply a high probability of G-protein-couples receptors (GPCRs) involvement in sensing salinity signals. It has clearly been indicated that GPCRs in association with G-proteins activate Ras-related small GTP-binding protein (Bhattacharya, Babwah & Ferguson, 2004). This process is followed by activation of IP₃ signaling pathway, Ca²⁺ production, Ca²⁺ pathway activation, and finally gene expression changes (Ghosh & Xu, 2014). In conjunction with the IP₃ pathway role in canola response to salt stress, it has been reported that high salinity induces some components of the IP₃ pathway. Transcriptomic analysis of the Brassica napus revealed that phosphatidylinositol-specific phospholipase C2 (BnPLC2), phosphatidylinositol 3-kinase (BnVPS34) and phosphatidylinositol synthase (BnPtdIns S1) have significantly differential expression under salt stress (Das et al., 2005). In the case of the Ca²⁺ pathway, annexin identification in canola root (Banaei-Asl et al., 2015; Yíldíz, Akçalí & Terzi, 2015) supports these pathway roles in sensing and signaling salt stress. The annexin mediator roles have been characterized in response to abiotic stresses as targets of the Ca²⁺ signaling pathway (Konopka-Postupolska et al., 2009). Further confirmation of the active role of the above-mentioned pathways in canola comes from identification of calcium-dependent protein kinase (CPK) differential expression under abiotic stresses, including salt stress (Zhang et al., 2014a). CPKs sense Ca²⁺ and act as a kinase.

Taken together, GPCRs and Ras-related small GTP-binding proteins are involved in salt stress perception, sending the message through the IP₃ signaling pathway. This is followed by high accumulation of Ca²⁺ ions in the cytosol, mainly through the action of calcium channels located at the surface of smooth endoplasmic reticulum. In response to high Ca²⁺ concentration, the Ca²⁺ pathway is activated and, subsequently, the salinity signals are passed to the nucleus where the alteration of the relevant genes occurs in response to the salinity signals.

Gene expression regulation

Three layers of gene expression regulation have been revealed in response to salinity in canola cultivars. However, many other mechanisms remain to be explored. The first level of gene expression regulation is at the transcription stage, which is mediated by transcription factors. Transcription factors are major players interacting with other proteins, especially RNA polymerases, and cis/trans acting elements on the regulatory regions of the genome. Lee et al. (2008) reported that 56 genes in canola encode putative transcription factors as factors which are altered under abiotic stresses. Among these genes, those that have been shown upregulation by more than 5-fold under salt stress, are from AP2-EREBP family (ATERF11, CBF4/DREB1D, CBF1/DREB1B, ATERF4/RAP2.5, DREB2A, CBF1/DREB1B, DREB2A, and ATERF11), Basic-Helix-Loop-Helix (bHLH) family (AtbHLH17), Basic region leucine zipper (bZIP) family (AtbZIP55/GBF3), C2H2 family (ZAT10, ZAT12/RHL41, ZAT6, and ZAT102/RHL41), Heat stress family (ATHSFA1E), Homeobox family (ATHB-7), NAC family (ANAC036, ANAC029/ATNAP, ANAC055/ATNAC3, ANAC047, ANAC072/RD26, ANAC002/ATAF1, ANAC019, and ANAC032), and WRKY family (ATWRKY53, ATWRKY40, and ATWRKY33). These transcription factors are induced in response to the salt-stress message transmitted by sensing and signaling molecules. Thereafter, complex gene regulatory networks consisting of transcription factors and other proteins govern the expression of numerous genes.

Epigenetic events are another mechanism for gene regulation that has been shown in canola. Epigenetic regulation of stress-responsive genes has been shown to play a pivotal role in the plant under various conditions (Chinnusamy & Zhu, 2009; Luo et al., 2012). In this context, it has been reported that when salinity is added to pretreated plants with osmotic stress, plants with histone modifications accumulated Na⁺ ion in a concentration that is not toxic for the plant (Sokol et al., 2007). In canola, DNA methylation and histone modification have been reported in response to salinity. When canola is exposed to salt stress, de novo methylation and demethylation events occur at CpCpGpG sites (Labra et al., 2004). The genes with epigenetic modifications are less known in canola. The ethylene-responsive element binding factor (EBF) is one of the genes that undergo DNA methylation in canola under salt stress (Guangyuan et al., 2007). In this regard, studies on canola are very scarce. In Arabidopsis and tobacco, it has been shown that, under salinity, histone proteins are rapidly upregulated and are phosphorylated, resulting in a low Na⁺ accumulation (Sokol et al., 2007). These results suggest the possible roles of DNA methylation/demethylation and chromatin (histone) modifications in regulating salt-responsive gene expression.

In the post-transcriptional stage, Micro RNA (miRNA) roles have been studied in salt-treated canola. The miRNAs are non-coding RNA ranging from 20 to 24 nucleotides in length. It has been reported that more than 340 miRNAs participate in the post-transcriptional regulation of the salt-responsive genes in canola (Jian et al., 2016). The miRNAs are negative regulators that bind their target gene transcript and prevent the gene from being translated. It has been indicated that miRNAs induced under a specific stress, mainly target transcription factors. One of the transcription factors that are demonstrated as being targeted by miRNAs, is the NAC transcription factor (Nakashima et al., 2012). Lee et al. (2008) reported that all the transcription factors belonging to NAC family show downregulation in canola exposed to salinity. Salt tolerance homolog2 (STH2) is another target of miRNAs under salt stress reported in canola (Lee et al., 2008).

Unfortunately, there are not studies enough to indicate comprehensive information about miRNAs and their targets under salinity stress in canola. However, studies on other plants under salinity stress, specifically Arabidopsis, have revealed that many transcription factors and genes, such as superoxide dismutase and Laccases multi-copper-containing glycoproteins, are under the control of miRNAs (Liang et al., 2006; Lu et al., 2013; Stief et al., 2014). The miRNA key roles in adaptation to different types of stresses and their implications in the plant growth and development have turned attention to the use of miRNAs as a new promising target for improving tolerance to harsh environments (Zhang, 2015).

Protein synthesis and modifications

The function of the protein synthesis machinery is to supply proteins needed for the cellular processes. The proper function of this machine is vital for plants under any kind of stress. Proteomic analysis of canola roots and leaves under salinity has identified several proteins related to protein synthesis and modifications. Generally, proteins are categorized into two group: the first consists of proteins related to the proteins synthesis machine, and the second of proteins related to the correct folding and stability of newly synthesized proteins. From the first group, ribosomal related proteins (only in leaves) were identified. Ribosomal proteins constitute the ribosome structure, functioning in translation. From the second group, Heat Shock Protein (HSP) families and ubiquitin protein (only in the leaf) were identified in canola (Banaei-Asl et al., 2015; Banaei-Asl et al., 2016; Bandehagh et al., 2011; Gharelo-Shokri et al., 2016; Yíldíz, Akçalí & Terzi, 2015). Decreased protein synthesis is a common observation under salt stress (Tuteja, 2007). However, studies on tolerant cultivars of canola have shown that protein synthesis was enhanced under salinity stress, especially for ribosomal proteins, heat shock proteins, and ubiquitin proteins (Banaei-Asl et al., 2015; Banaei-Asl et al., 2016; Bandehagh et al., 2011). It seems that canola’s strategies in the root and leaves are different. In the root, only the differential expression of heat shock proteins, Hsp 70, has been reported (Banaei-Asl et al., 2016; Bandehagh et al., 2011), while in the leaves, ribosomal, heat shock, and ubiquitin proteins have been upregulated (Banaei-Asl et al., 2015). Hsp 70 is a chaperone protecting newly synthesized proteins from aggregation as well as ensuring proper folding. Their high accumulation is induced by many environmental stresses, such as heat, drought, salinity, cold, and wound healing (Boston, Viitanen & Vierling, 1996; Burdon, 1988). Similar to Hsp 70, ubiquitin acts as a regulator to stabilize the functions and location of the proteins through covalently binding those proteins at specific sites (Dametto et al., 2015).

Dynamic changes of canola genes and proteins

Several transcriptomic and proteomic studies performed on canola under salt stress indicating that DEGs and differentially expressed proteins in both leaves and roots are mainly categorized into seven functional groups, except genes/proteins related to signaling, transcription, protein synthesis and modifications described in the earlier sections (Banaei-Asl et al., 2015; Banaei-Asl et al., 2016; Bandehagh et al., 2011; Deinlein et al., 2014; Gharelo-Shokri et al., 2016; Jia et al., 2015; Yíldíz, Akçalí & Terzi, 2015). According to the number of genes/proteins identified in each functional group, these groups are (a) carbohydrate and energy metabolism, (b) stress and defense, (c, d) metabolism and photosynthesis (in the case of leaves), (e) cell structure, (f) membrane and transport, and (g) cell division, differentiation and fate. These groups have different members in number but they are respectively mentioned from high to low number (Deinlein et al., 2014). In canola roots subjected to stress, the number of proteins related to carbohydrate and energy metabolism are more than in other functional groups. However, proteins related to amino acid metabolism and cell structure are also remarkable in abundance. In carbohydrate and energy metabolism the majority of proteins are from the TCA cycle, electron transport chain (ETC), and glycolysis (Banaei-Asl et al., 2015; Gharelo & Bandehagh, 2017). In canola leaves, the high abundance functional proteins belong to photosynthesis, protein synthesis and degradation, amino acid metabolism, and damage repair and defense response (stress and defense) (Banaei-Asl et al., 2016; Gharelo-Shokri et al., 2016; Jia et al., 2015). In the photosynthesis-related, differential abundance of chlorophyll a/b binding protein, chloroplast RuBisCO activase, ribulose bisphosphate carboxylase (RuBisCO) small and large subunit, and ribulose bisphosphate carboxylase/oxygenase have been reported in salinity-tolerant canola cultivars (Banaei-Asl et al., 2016; Bandehagh et al., 2011; Yíldíz, Akçalí & Terzi, 2015). Because of the importance of membrane transporter proteins, stress and defense, and amino acid metabolism for salt tolerance, we described the proteins and genes identified in these functional groups at forthcoming in a subsequent section in detail. Here, it is worth mentioning two frequently observed proteins, actin, and tubulin, identified in canola root and leaves (Banaei-Asl et al., 2016; Bandehagh et al., 2011; Jia et al., 2015; Yíldíz, Akçalí & Terzi, 2015). Similar to other plant response to salt conditions (Agarwal et al., 2013; Bandehagh et al., 2011; Gharelo-Shokri et al., 2016; Ghosh & Xu, 2014; Gupta & Huang, 2014; Liang et al., 2018; Mickelbart, Hasegawa & Bailey-Serres, 2015; Parihar et al., 2015; Tuteja, 2007; Wan et al., 2017; Yíldíz, Akçalí & Terzi, 2015; Zhang et al., 2011), it seems that canola alters its cytoskeleton basic components (i.e., actin and tubulin) under salt stress. It has been demonstrated that cytoskeleton dynamic remolding is linked to some of the main transmembrane transports, such as K⁺ channel (Ahanger et al., 2017; Katz et al., 2007; Tuteja, 2007). Another important point relating to the functional category of differentially changed proteins is the unknown proteins that constitute about 1% to 20% of total differentially changed proteins in each study results, especially in studies on the root (Banaei-Asl et al., 2015; Banaei-Asl et al., 2016; Bandehagh et al., 2011; Gharelo-Shokri et al., 2016; Jia et al., 2015; Toorchi & Kholgi, 2014; Yíldíz, Akçalí & Terzi, 2015; Zhang et al., 2011). Identification of these proteins could provide more insights on salt-response mechanisms.

In each functional category, the abundance of genes/proteins dynamically changes on the basis of duration and severity of salt stress, organ, and even between different leaves. Bandehagh et al. (2011) reported that when salinity severity increases from 175 to 350 mM NaCl, the number of differentially expressed proteins increase. They compared the increases between second leaf (young leaves) and third leaf (older leaves) and conclude that the younger leaves show significantly more increase in their salt-responsive proteins. Another study, by Hu et al. (2013), indicated that the expression levels of BnBDC1, BnLEA4, BnMPK3, and BnNAC2 are upregulated 1 h after salt stress, while their expression is downregulated 3, 6, and 12 h after the stress. Jia et al. (2015) in their study on the dynamic changes of canola’s proteome under 200 mM NaCl at three-time points (24 h, 48 h, and 72 h) indicated that the salt-responsive proteins have a dynamic pattern. They explained the dynamic behavior of canola proteome by clustering the salt-responsive proteins into two main clusters. In the first cluster, the proteins were grouped into two sub-groups, A and B. The sub-group A showed downregulation at 24 h after salinity treatment, while these proteins (sub-group A) were upregulated at 48 h and 72 h time points. These studies indicate the importance of dynamic analysis in understanding molecular mechanisms. Investigation of the different organs, different time points, and different severities of salt stress as well as integrated transcriptomic and proteomic dynamics could provide deep insights into the response of canola to salinity.

Proline synthesis

Studies on canola have indicated significantly increased proline contents in the root and leaves of both salt-tolerant and sensitive cultivars under salt stress, and comparatively more in tolerant cultivars than sensitive ones (Dolatabadian, Sanavy & Chashmi, 2008; Xue, Liu & Hua, 2009). Proteomic analysis of canola-tolerant cultivars indicated that the abundance of P5CS in the root and leaves are increased (Banaei-Asl et al., 2015; Banaei-Asl et al., 2016; Yíldíz, Akçalí & Terzi, 2015). It has been shown that high accumulation of proline in canola attributes to activating its biosynthesis and preventing its degradation (Xue, Liu & Hua, 2009). This was confirmed in another study by Madan et al. (1995) in which they reported that the activity of proline synthesis enzymes, pyrroline-5-carboxylate reductase and ornithine aminotransferase, increases about three-fold, whereas the proline-degrading enzyme, proline oxidase, was decreased by salt stress. These findings confirm that canola changes proline metabolism in tune with the increase of proline contents under salt stress. Proline acts as an osmolyte, ROS scavenger, redox buffer, and molecular chaperones under stressful conditions. This suggested that, during the recovery phase, proline serves as a signaling molecule for cell growth, proliferation, and death (Deinlein et al., 2014; Liang et al., 2018; Xue, Liu & Hua, 2009).

Alternative regulation of ion transporter proteins in the root and leaf

H⁺/K⁺ transporters (HKT) and Na⁺/K⁺ co-transport show downregulation in canola root but upregulation in the leaves during salt stress (Yong et al., 2014). This pattern of HKT expression is to reduce Na⁺ uptake through the root from rhizosphere and to protect the leaf against Na⁺ accumulation by removing Na⁺ from xylem sap into parenchymal cells. These type of ion transporters are responsible for Na⁺ uptake into the root and leaf cells. In Arabidopsis, it has been reported that mutants with HKT deficiency are salt hypersensitive with a high amount of Na⁺ in leaves and a low amount in the root (Mäser et al., 2002). Furthermore, the role of HKTs has been indicated in removing Na⁺ ions from xylem sap into parenchymal cells located around xylem (Berthomieu et al., 2003).

Three transporters involved in transporting Ca²⁺ into the cytosol, including CNGCs (cyclic nucleotide-gated ion channel), GLRs (glutamate receptor), and ACAs (ATPase homologous to Arabidopsis), have been shown to be upregulated both in canola root and leaf, while CAXs (endosomal cation/proton exchanger), which is involved in removing Ca²⁺ from the cytosol into the vacuole, is downregulated in the root (Yong et al., 2014). It seems that overall actions of these ion transporters are to increase Ca²⁺ ions in the cytosol. High levels of Ca²⁺ could have two main consequences for the cell. First, Ca²⁺ ions accumulated in the cytosol activate Ca²⁺ sensor proteins of the cytosol to signal the presence of stress (Knight, Trewavas & Knight, 1997). Second, these ion channel actions in transporting Ca²⁺ ions also contribute to K⁺ uptake which is important for maintaining the cellular hemostasis (Ali, Zielinski & Berkowitz, 2005).

Canola upregulates ion channels responsible for K⁺ influx into the cytosol from vacuole (KCOs) and from stele into xylem (SKORs) (Labra et al., 2004). However, it downregulates K⁺ ion channels functioning in the efflux of K⁺ out of the cell (KEA) (Yong et al., 2014). It has been illustrated that K⁺ functions in controlling whole plant ion hemostasis and the cell turgor (Shabala & Cuin, 2008; Su et al., 2001). In tune with these changes in K⁺ transporters, PHT and V-ATPase are upregulated by canola (Yong et al., 2014). These proteins act to provide the driving force to maintain Na⁺ at a low level and K⁺ in a high level in the cell cytosol (Zhu, 2003).

Reactive oxygen species scavenging system

For ROS production, there are two conflicting views: while many reports blame ROSs for attacking major components of the cell and for interfering with many vital metabolic pathways, some reports do not know ROS production necessarily as a symptom of these dysfunctions, but place emphasis on ROS signaling roles under abiotic stress. However, all studies have found the efficiency of the plant ROS scavenging system to be one of the main stress-tolerant traits. Regarding this, a significantly high activity of APX and CAT is reported for a canola-tolerant cultivar (i.e., Hyola308) treated with 200 and 300 mM NaCl (Heidari, 2010). Similarly, another study reported high activity of SOD, GPX, and CAT as well as upregulation of Cu/Zn SOD in the leaves under 150 mM NaCl. Upregulation of Cu/Zn SOD has similarly been demonstrated for Hyola308 under 150 and 300 mM NaCl stress (Banaei-Asl et al., 2016). All these studies provide evidence to confirm that tolerant cultivars show remarkably more ROS scavenging activity than sensitive cultivars (Banaei-Asl et al., 2016; Heidari, 2010; Yíldíz, Akçalí & Terzi, 2015). Furthermore, the role of glutathione synthesis in canola is defined as oxidative protective mechanism (Ruiz & Blumwald, 2002). In this context, cysteine and glutathione content was measured for wild-type and salt-tolerant transgenic canola, transformed with vacuolar Na⁺/H⁺ antiporter from Arabidopsis, under 150 mM NaCl. After 15 days of continuous salt stress, cysteine and glutathione content were increased three-fold in wild compared to tolerant canola. This observation confirms the possible protective role of glutathione synthesis in coping with oxidative damages.

Candidate genes/proteins for canola tolerance

Several studies have tried to identify the main gene(s)/protein(s) responsible for canola tolerance. Knowledge of these key components among the complexity of the salt response networks is a critical step toward engineering salt-tolerant canola. Gharelo-Shokri et al. (2016) in their study reported six hub genes in tolerant cultivars, including UDP-glucose dehydrogenase, Methionine synthase, Malate dehydrogenase, Triose phosphate isomerase, heat shock protein 70, and Fructose-bisphosphate aldolase in constructed protein-protein interaction network of canola salt-induced proteins. Hub genes are high interactive elements of the constructed network that is regarded as the main components of the network (Ning et al., 2010). Furthermore, some of the candidate genes/proteins for canola salt tolerance could be extracted from studies about an external application of materials that promote canola tolerance under salinity. In this respect, Banaei-Asl et al. (2015) found that, in response to plant growth promoting rhizobaceria (PGPR) inoculation, canola root upregulates glyceraldehyde-3-phosphate dehydrogenase and downregulates S-adenosylmethionine synthase, aldehyde dehydrogenase, and malate dehydrogenase under 150 and 300 mM of NaCl. Their study demonstrated that inoculated plants show significantly more root length, root dry weight, high K⁺ levels, and a low Na⁺ and Cl⁻ levels compared to non-inoculated plants. They showed that the better growth parameters of the inoculated root are due to differential abundant bacteria-responsive proteins. Moreover, in another study, they indicated that bacterial inoculation gives canola more tolerance through an increased abundance of proteins related to glycolysis, TCA, and amino acid metabolism, especially succinate dehydrogenase (Banaei-Asl et al., 2016).

In canola, several studies have shown that overexpression of some genes results in altered salt tolerance. One example is ectopic overexpression of Dehydration-Responsive Element Binding transcription factors (DREBs). Plants transformed for high expression of DREBs increase their salt-responsive gene expression including COR14, HSF3, HSP70, PEROX and RD20 showing more tolerance. According to these results, transgenic plants are able to survive under the salinity level in which wild-type plants are more sensitive (Shafeinie et al., 2014). Given that 5-Aminolevulinic acid (5-ALA) exogenous application resulted in salt tolerance in treated plants, Sun et al. (2015) transformed canola with 5-ALA-encoding gene, YHem1, and studied the growth of the transgenic canola capable to synthesize more 5-ALA, and wild-type canola under salinity stress. They reported that under both short-term and long-term salinity, transgenic canola show more yield, more chlorophyll content, high activity of antioxidant enzymes, high proline content, high sugar content, and more free amino acids compared to wild-type canola. Furthermore, they demonstrated that an increased tolerance of transgenic canola could be related to the upregulation of Rubisco small subunit and significantly high levels of Fe metal. In contrast to these studies, in which increased tolerance has been reported, it has been suggested that expression of Brassica napus TTG2 causes sensitivity to salt stress through downregulation of the genes TRYPTOPHAN BIOSYNTHESIS 5 (TRP5) and YUCCA2 (YUC2) encoding indole-3-acetic acid (IAA), hence, declining the endogenous IAA content. It is expected that in future research in transgenic plants, the newly emerging CRISPR/Cas9 system will provide more information about molecular components responding to salt stress (Osakabe et al., 2016).