The results presented in this paper show that lipophilic components of A. nodosum extracts (LPC) induced specific biochemical changes leading to enhanced tolerance to freezing stress in Arabidopsis thaliana. LPC-treatment of the roots resulted in enhanced freezing tolerance in the aerial parts of the plant, which implicates LPC in inducing systemic metabolic responses.
Proline is one of several compounds with roles as compatible solutes that accumulate in response to freezing and drought stress,  and osmotic stress . Our results on the quantification of proline accumulation in wild-type and LPC treated Arabidopsis plants showed that application of ANE or LPC, resulted in a 50% increase in free proline in response to freezing, as compared to control plants.
In higher plants, proline is synthesized via the glutamate pathway or the ornithine pathway [18, 22, 33]. The glutamate pathway is predominant during abiotic stress and under nitrogen limiting conditions [33, 34], while the ornithine pathway takes effect under nitrogen abundance . Plants accumulate proline in response to freezing-induced osmotic stress by simultaneous activation of proline biosynthesis (via the P5CS1 pathway) and/or down regulation of proline degradation (via the ProDH pathway) . The reciprocal regulation of the Δ1-pyrroline-5-carboxylate synthetase (P5CS), a rate-limiting enzyme in proline biosynthesis, and proline dehydrogenase (ProDH), responsible for proline degradation, is a key mechanism in the control of cytosolic proline concentration .
The results of our experiments with p5cs1 mutant plants confirmed the role of proline in ANE-mediated freezing tolerance, in that the p5cs1 mutants, treated with ANE, LPC and the water control, all showed similar freezing damage. These observations were in agreement with the results of the gene expression studies. Results of the gene expression analysis suggested that increased proline concentration in the LPC-treated plants was a result of a coordinated increase in P5CS1 and P5CS2 transcripts and, in part, due to decreased in ProDH.
Several studies have suggested that cold-induced sugar accumulation enhances the degree of plant freezing tolerance. In Arabidopsis, a large increase in the degree of freezing tolerance is positively correlated with soluble sugar content [36, 37]. Similarly, increased sucrose levels in transgenic Arabidopsis plants, over-expressing a gene for sucrose phosphate synthase, equated with freezing tolerance . Conversely, sensitive to freezing 4 (sfr4) mutants exhibited an impaired freezing tolerance response due to a reduced accumulation of sugar levels at low temperature relative to the wild type [39, 40]. In LPC-treated plants, increased accumulation of soluble carbohydrates was brought about by multiple mechanisms such as inducing polysaccharide degradation (i.e. starch, galactose etc.), promoting the biosynthesis of soluble carbohydrates (i.e. glucose, fructose, Raffinose/stachyose), and inactivating the sucrose degradation pathways.
Recently, Uemura et al.  showed that the sensitivity of the Arabidopsis mutant sfr4 to freezing was due to its low sugar content, as manifested by loss of osmotic responsiveness. Additionally, it has been demonstrated that exogenous sucrose, at low concentrations, serves as a substrate for low temperature-induced metabolic alterations, while at higher concentrations it has a direct cryoprotective effect on cellular membranes . Although an increased concentration of cytosolic soluble sugar has been observed in response to freezing-induced osmotic stress, in many plant species , it is unclear whether the accumulated sugars act as osmolytes, or serve as a source of energy and carbon, that fuels the metabolic changes leading to enhanced freezing tolerance .
A positive correlation between sugar accumulation and freezing tolerance has been widely documented in many plant species, including Arabidopsis [36, 43]. In the present study, application of ANE or LPC resulted in a significantly enhanced accumulation of total soluble sugars in response to freezing, as compared to untreated controls. The results were further strengthened by analyzing the sensitivity of sfr4 mutant plants to freezing.
1H NMR analysis of the Arabidopsis metabolome, during freezing, revealed that the application of LPC altered biochemical pathways resulting in the accumulation of specific metabolites, leading to enhanced freezing tolerance. The 1H NMR spectra of plants treated with LPC was dominated by peaks with chemical shifts (δ) around 0.8 to 4.0 ppm. These chemical shifts primarily represented 2 major groups of compounds; 0.8 – 2.8 ppm represents lipophilic components like fatty acids and sterols, while resonance at 3.0 to 4.0 ppm represented carbohydrates, sugars, sugar alcohols and organic acids . Our results are in agreement with previous reports. These findings showed that multiple primary metabolites could act collectively, as compatible solutes, ameliorating the osmotic stress caused by freezing.
1H NMR peaks with chemical shifts (δ) around 0.8 to 2.8 ppm, represented the lipophilic components such as fatty acids and sterols, were another major group of metabolites which showed specific changes during freezing stress in LPC-treated plants. Fatty acids are major components of cellular membranes, suberin, and cutin waxes that provide structural barriers to the environment. At low temperature, plant membranes undergo a transition from a liquid crystalline state to a gel-like phase with reduced fluidity contributing to ion leakage and deactivation of membrane proteins . Fatty acids contribute to inducible stress resistance through the remodeling of membrane fluidity . The ability to adjust membrane fluidity by modulating levels of unsaturated fatty acids is a feature of freezing-tolerant plants. Fatty acid unsaturation is thought to reduce the propensity of cellular membranes to undergo freezing-induced, non-bilayer phase formation, thus enhancing membrane integrity and cellular function during freezing .
Metabolite profiling revealed an increase in unsaturated fatty acids in plants treated with LPC when exposed to freezing. The spectral peaks (or peak groups) at δ = 5.4-5.2 ppm (protons on double bond carbons), 2.8, 2.3, 2.1, 1.6, 1.4-1.2 ppm (protons on alkyl chain), and 1.0-0.8 ppm (terminal methyl group of alkyl chain) are indicative of unsaturation of fatty acid. We have previously reported that application of ANE or LPC resulted in less tissue damage and electrolyte leakage, as compared to controls, thereby improving the LT50 values . The metabolite profiles suggested that that LPC-mediated freezing tolerance in Arabidopsis may be the result of a combination of metabolic adjustments and increased fatty acid content in Arabidopsis.
Chemical components in the extracts of A. nodosum caused a rapid biochemical response leading to an increased accumulation of osmoprotectants (proline and soluble sugars) and unsaturated fatty acids. The results of the present study suggested that application of these extracts elicited responses reminiscent of a priming effect. Priming is a phenomenon whereby previous exposure to biotic or abiotic stress stimuli makes a plant more resistant to future incidents . Chemical priming is a novel strategy, wherein application of certain chemicals mimic moderate stress stimuli, through physiological and/or hormonal reactions and brings about a priming reaction in the target plants. Exogenous application of chemicals inducing a priming effect, in abiotic stress tolerance has been previously reported . Application of a non-protein amino acid, β-aminobutyric acid (BABA), was shown to induce a priming response in Arabidopsis towards salt stress tolerance (through induction of ABA-dependant elements such as RAB18and RD29A) and salicylic acid-dependent disease tolerance (through PR-1, PR-5 response) . The results presented in this paper suggest the possibility of a chemical priming effect in Arabidopsis, as a reaction to the application of ANE or LPC, resulting in enhanced freezing tolerance.
Further, we used a whole-genome approach to determine the ANE-mediated freezing tolerance in Arabidopsis plants. Application of LPC significantly altered gene expression for 1% of the Arabidopsis genes. In this study, we focused on gene expression related to three aspects: (1) response to stress and/or stress stimulus; (2) compatible osmolyte accumulation; (3) changes in membrane lipid profile in response to stress.
Around 40 annotated genes were found to be up-regulated in our focus category with four genes (At1g10760, At1g55920, At1g09780, At3g22840) found to be directly involved in low temperature stress tolerance in Arabidopsis [48–51]. Another major group of induced genes was those expressed in response to osmotic stress (like water deprivation, salt stress etc.). The ability to survive periods of desiccation is an important adaptation to freezing temperatures and many of the ‘water-deprivation controlled’ genes are also required for maximum survival during freezing stress . Many of the genes expressed during cold acclimation are also inducible by drought stress, and are likely to play a role in protection against cellular dehydration, which occurs during both freezing and drought conditions . This finding is consistent with previous observations, that those plants which are able to efficiently manage osmotic stress, are more tolerant to freezing temperatures .
Genes activated in response to biotic stress and/or stimuli were also activated owing to the fact that both biotic and abiotic stress stimuli share a common signaling cascade to bring about physiological responses . For example, salicylic acid (SA) has long been known as a signal molecule in the induction of defense mechanisms in plants [56–58]. Exogenous application of SA has been shown to improve freezing tolerance in wheat by regulating the ice nucleation activity of apoplastic proteins [59, 60]. Ding et al.  have shown that 0.01 mM methyl salicylate and methyl jasmonate treatment improved the cold tolerance of tomato fruits. Moreover, SA was shown to accumulate during low temperatures in chilling-resistant Arabidopsis plants .
Similarly, several studies have suggested that cold-induced sugar accumulation enhances the degree of plant freezing tolerance. In Arabidopsis, a large increase in the degree of freezing tolerance is positively correlated with soluble sugar content [36, 37]. Increased sucrose levels in transgenic Arabidopsis plants over-expressing a gene for sucrose phosphate synthase equated with freezing tolerance . Conversely, sensitive to freezing 4 (sfr4) mutants exhibited an impaired freezing tolerance response due to a reduced accumulation of sugar levels at low temperature, relative to the wild type [39, 40]. In LPC-treated plants, increased accumulation of soluble carbohydrates was brought about by multiple mechanisms such as induction of polysaccharide degradation (i.e. starch, galactose etc.), promoting biosynthesis of soluble carbohydrates (i.e. glucose, fructose, raffinose/stachyose) and inactivating the sucrose degradation pathways.
Starch is the main carbohydrate store in plants. Regulation of starch metabolism, in particular in response to environmental cues, is of primary importance for carbon and energy flow in plants . Along with photosynthesis, starch degradation also plays a significant role in cold-induced sugar accumulation and enhanced freezing tolerance in Arabidopsis [43, 48, 64]. Starch-related glucan/water dikinases encoded by the Arabidopsis ‘Starch Excess’ 1 and 4 genes (SEX1, SEX4), regulate starch degradation in plastids by phosphorylating starch, thereby ensuring better accessibility by starch-degrading enzymes during cold induced starch degradation. SEX1 plays an essential role in the cold-induced starch degradation, sugar accumulation, and freezing tolerance enhancement during an early phase of cold acclimation . SEX4/DSP4 phosphatase activity has been shown to be regulated by the redox state  and unfavourable environmental stress conditions alter the redox balance within the cells. It is believed that SEX4/DSP4 phosphatase activity may be regulated in response to environmental stress and might be associated to MsK4/AtK-1, a plastid-localized protein kinase associated with starch granules, which is an important regulator that adjusts carbohydrate metabolism during environmental stress . SEX 1 and SEX 4 were found to be activated in LPC-treated plants during freezing stress, as compared to control plants.
Raffinose family oligosaccharides (RFOs) such as raffinose and stachyose are accumulated during the process of cold acclimation, when plants acquire increased frost tolerance [66–68]. Arabidopsis plants with higher rates of raffinose biosynthesis demonstrated increased accumulation of raffinose and galactinol upon cold acclimation and exhibited higher freezing tolerance . Molecular mechanisms by which RFOs influence cellular freezing tolerance are not clear, but it has been shown previously that raffinose can stabilize isolated chloroplast thylakoid membranes during a freeze–thaw cycle . Galactinol synthase (GolS) catalyses the first committed step in the biosynthesis of RFOs  and, therefore its expression, provides an experimental tool to assess the level of RFOs during freezing stress, to analyze the function of RFOs as osmoprotectants . LPC application stimulated the raffinose/stachyose biosynthetic genes GolS2 and GolS3 during freezing.
LPC treatment specifically induced a major pathway involved in galactolipid biosynthesis and lipid trafficking in thylakoid membranes controlled by the gene DGD1. Other changes include down-regulation of sterol/cholesterol biosynthesis, regulation of Acyl-CoA thioesterases and down regulation of long chain fatty acid synthesis (Figure 8).
Membranes are the major injury sites during freezing stress [71, 72], and membrane lipids undergo substantial changes when plants are exposed to freezing temperatures . It has been well established that membrane polar lipid composition is one of the important factors controlling the structure and efficiency of thylakoid membranes via specific lipid–protein interactions and/or the dynamic properties of the lipid bilayer [74, 75]. The galactolipids constitute the bulk (close to 80%) of the thylakoid lipid matrix and, within green plant parts, 70 to 80% of the lipids are associated with photosynthetic membranes. During freezing, dramatic alterations take place in plastid membranes, decreasing the monogalactosyldiacylglycerol (MGDG) content and increasing digalactosyldiacylglycerol (DGDG) content [76, 77]. The ratio of DGDG to MGDG is critical for correct protein folding, insertion and intracellular protein trafficking in the chloroplast  during temperature stress . DGD1 (digalactosyldiacylglycerol synthase 1) is the enzyme involved in the conversion of MGDG to DGDG in photosynthetic membranes of the chloroplast . The dgd1 mutant of Arabidopsis was impaired in galactolipid assembly, as suggested by a 90% reduction in digalactosyl lipid content . DGD1 (digalactosyldiacylglycerol synthase 1) was found to be highly expressed in LPC-treated plants during freezing stress. Taken together, it may be concluded that activation of digalactosyldiacylglycerol synthase 1 (DGD1) resulted in efficient conversion of MGDG to DGDG, thereby maintaining improved stability of membranes and reduced ion leakage in LPC treated plants.
An early response to low temperature stimulus, by tolerant plant species like winter barley oats, is decreased in membrane fluidity . A major adaptation among the winter tolerant species is the ability to maintain the fluidity of membranes by reducing the ratio of free sterol to total phospholipids. This is achieved by a decrease in the free sterols and an increase in the proportion of phospholipids . Sterol/cholesterol biosynthesis was found to be highly down regulated in LPC-treated plants during freezing stress. A further modification observed in the lipid metabolism of LPC treated plants was the regulation of Acyl-CoA thioesterase.