Plant Prenyllipid Antioxidants and Their Role in the Response to Cold Stress

1. Introduction

Being sessile organisms, plants have to deal with the wide variety of unfavourable and unavoidable environmental conditions, such as drought, high light, extreme temperatures, waterlogging, salinity, and many others. These stresses pose a threat to plant survival and reproduction, they also limit growth and yield. Among them, low temperature is one of the most harmful to higher plants [1,2].

Exposure to environmental stresses usually leads to the excessive formation of reactive oxygen species (ROS), as a result of the disturbance of various metabolic processes [3]. The situation in which ROS generation in the cell exceeds the ability of their detoxication is called oxidative stress. Plants are especially prone to oxidative damage because (1) they perform oxygenic photosynthesis and therefore produce additional O₂ in their green tissues (2) they contain a lot of chlorophyll (Chl) that can act as photosensitiser and transfer its excitation energy to O₂ (3) they have a photosynthetic electron transfer chain from which electrons can leak to O₂ [4]. If the photosynthetic apparatus absorbs more energy that can be used for beneficial reactions of photosynthesis, excess energy fuels the unwanted side reactions leading to enhanced ROS formation. In low temperatures, the chemical reactions of photosynthesis are slowed down, whereas the absorption of light by the photosynthetic apparatus is quite efficient, which results in a decrease of the threshold above which certain intensity of light becomes excessive [4,5]. Furthermore, among the mechanisms of acclimatisation to cold stress, an important one is the maintenance of membrane fluidity, necessary for the proper functioning of cellular metabolism, in particular photosynthetic and respiratory electron transfer chains, as well as proper membrane transport and membrane-related signalling. To achieve this objective, many organisms enhance the share of polyunsaturated fatty acid chains (PUFA) in their membrane lipids [6]. However, PUFA are very susceptible to oxidation [7]. Therefore, antioxidant protection of lipids is crucial to provide tolerance to cold stress. Prenyllipid antioxidants, such as carotenoids, and isoprenoid chromanols and quinols, are potent lipophilic antioxidants occurring in membranes and lipid storage sites [8]. Carotenoids are also crucial for safe dissipation of excess light energy absorbed by the photosynthetic apparatus [9]. Additionally, the compounds mentioned above participate, directly or indirectly, in signalling during stress acclimatisation [4,9].

In this review we tackle the role of lipophilic antioxidants in low temperature-induced stress. The first subchapter focusses on ROS, their properties and harmful action, the sites of their formation and the ways of their detoxication. The second chapter is devoted to prenyllipid antioxidants, their chemical structures, occurrence, biosynthesis and function. Particular emphasis was placed on the compounds present in chloroplasts, because these organelles are the major site of ROS production in plant green tissues. The third chapter is about the impact of low temperature on plants, while the fourth summarises the literature data concerning the role of prenyllipid antioxidants in the response to cold stress.

2. Reactive oxygen species, their sites of formation in plants, and their action on biomolecules

Molecular oxygen appeared on Earth as a product of oxygenic photosynthesis. This crucial metabolic process has evolved in ancient cyanobacteria and later was passed onto photosynthetic eukaryotes, along with the acquisition of plastids, organelles of direct or indirect cyanobacterial origin [10,11]. The presence of O₂ in the atmosphere and waters enabled the subsequent evolution of energetically efficient aerobic respiration [12]. However, aerobic metabolism has its dark side, which is the formation of ROS as its by-products [13].

ROS consist of radical and non-radical forms. Molecular oxygen in its ground state is diradical that contains two unpaired electrons with parallel spins in two antibonding orbitals π*2p. Such a form is called triplet oxygen (³O₂). This unusual electron configuration makes ³O₂ less reactive than one could imply on the physicochemical characteristics of oxygen atoms [13]. Excitation of ³O₂ results in spin reversal of one of the unpaired electrons, leading to the formation of singlet oxygen (¹O₂). Singlet oxygen can have electrons of opposite spins either in separate orbitals (very short-lived form, ¹Σ_g⁺O₂) or the same orbital (form ¹Δ_gO₂ of lower energy and longer lifetime). Paired electrons make ¹O₂ much more reactive than ³O₂ because the spin restriction is removed [14]. Singlet oxygen is thought to be the major ROS responsible for light-induced loss of photosystem II (PS II) activity and leaf damage [14]. In addition to excitation, ³O₂ can also undergo reduction. The complete four-electron reduction leads to the formation of water, while all its intermediates belong to ROS [15]. Among them, superoxide radical (O₂^∙−), is often formed as a result of electron leakage from the electron transfer chains. In low pH, O₂^∙− undergoes protonation [3,15]. Dismutation of O₂^∙−/HO₂^∙ leads to the generation of H₂O₂, which is less reactive, but may react with free metal ions, resulting in the formation of hydroxyl radical (OH^∙). Metal ions tend to bind to the surface of DNA and proteins. In such a case, the most dangerous type of ROS would be formed in the vicinity of the targets of its damaging action [16]. The most important types of ROS and their reactivity are shown in Figure 1. Other ROS worth mentioning are ozone (O₃), alkoxy radicals (RO^∙, where R is organic residue, for example fatty acid chain), peroxy radicals (ROO^∙) and hydroperoxides (ROOH) [16].

Being prone to react with organic molecules, especially those containing unsaturated bonds, and with redox-active metal ions, ROS can disturb cell biochemistry and structure [3]. The most important biomolecules vulnerable to ROS and the results of their oxidative damage are shown in Figure 2. Taking into account the genetic material, mitochondrial and plastid DNA is more endangered than that present in the nucleus, due to the relatively high production of ROS in these organelles (see below) and the fact that, unlike nuclear DNA, it is not associated with histones [16]. Among lipids, PUFA residues are particularly susceptible to lipid peroxidation [17]. Such residues, when present in phospholipids, provide sufficient membrane fluidity; therefore, they are abundant in membranes, where electron transfer chains occur. Unfortunately, these electron transfer chains are important sources of ROS. Lipid peroxidation can occur through direct oxidation of lipids by ¹O₂ or through a radical-mediated process, which can be initiated by HO₂^∙, OH^∙, NO₂^∙, perferryl radicals, carbon-centered radicals (R^∙), RO^∙, and ROO^∙. Because radical-mediated peroxidation is a self-propagating chain reaction, low amounts of radical initiators may ultimately cause extensive damage [16]. However, the analysis of specific lipid peroxidation products showed that more than 80% of nonenzymatic peroxidation in the leaves of higher plants is caused by ¹O₂ [18]. In addition to the compounds mentioned in Figure 2, ROS are able to oxidise other compounds, such as carbohydrates and pigments [3,19].

In photosynthetic cells, ROS are usually formed as a result of electron leakage from electron transfer chains and the photosensitising action of Chls; they can also be products of some enzymatic reactions (Figure 3). The most important sites of ROS production are chloroplasts, mitochondria, and peroxisomes [20]. In green tissues under light, the highest amounts of ROS are formed in chloroplasts, due to the occurrence of Chl-binding proteins, the functioning of the photosynthetic electron transport chain, and the increased concentration of O₂ because of its production at PS II [3]. Over-reduction of photosynthetic electron transfer chain leads to enhanced electron leakage and resulting formation of O₂^∙- . The electrons leak mainly from the Fe-S clusters of Photosystem I (PS I) and reduced ferredoxin, however O₂^∙- can be also formed at receptor side of PS II or within the pool of PQ [3,15,21]. Excessive reduction of photosynthetic chain elements also increases the risk of the formation of triplet excited states of Chl, which can transfer their excitation energy to ³O₂, leading to the generation of ¹O₂ [22]. In heterotrophic eukaryotes, mitochondria are the dominant sites of cellular ROS production. In photosynthetic eukaryotes, these organelles are thought to produce ROS less abundantly than chloroplasts. For example, in C₃ plants, H₂O₂ production in mitochondria is estimated to be 20 times lower than its production in chloroplasts. However, mitochondrial share in overall cellular ROS generation in plants is believed to be significant in darkness or in non-green tissues [3,23]. The major process responsible for mitochondrial ROS production is electron leakage from the respiratory electron transfer chain, mainly at complexes I and III; however, complex II and the over-reduced ubiquinone pool also play a role [23,24]. Some mitochondrial enzymes not involved in respiration are also known to produce ROS, but their contribution to mitochondrial ROS formation is minor [25]. In peroxisomes, H₂O₂ and O₂^∙- are generated by various catabolic enzymes. H₂O₂ is also a product of glycolate oxidase that participates in the photorespiratory pathway [26]. The ROS mentioned above can also be formed enzymatically at many other sites in the plant cell, which plays different roles, such as signalling, cell wall lignification, or response to pathogen [16].

To defend themselves against ROS, organisms have evolved various mechanisms based on low-molecular-weight antioxidants and enzymes (Figure 4). The former are able to quench ¹O₂ and scavenge various ROS; and can be subdivided into hydrophilic and hydrophobic ones. Lipophilic antioxidants belong to the prenyllipid class of compounds. They are crucial for the protection of cell membranes and lipid storage sites [4]. In addition to direct ROS scavenging, some low-molecular-weight antioxidants, such as ascorbate, glutathione, and some phenolic compounds are electron-providing co-substrates of ROS-detoxifying enzymes [27]. When enzymes are considered, crucial ones can directly detoxify certain types of ROS. Other enzymes participate in the recycling of low-molecular-weight antioxidants, oxidised as a result of ROS scavenging. There are also many enzymes responsible for the repair of oxidised biomolecules, especially proteins and nucleic acids. Glutathione-S-transferases, not shown in Figure 4, are a class of enzymes, members of which participate in various processes, including ROS detoxification, ascorbate recycling, and prevention of ROS formation due to detoxification of xenobiotics [3,16,27]. As plants are especially prone to oxidative stress, they have evolved a wide range of highly redundant antioxidant mechanisms [3]. Aerobic organisms have also evolved to use ROS for their benefit, in cellular signalling and to fight with pathogens [20]. Because various stress factors lead to the enhancement of ROS production in cells, these ROS may serve as signals, triggering plant responses [28]. Stress-induced ROS are known to participate in signalling pathways associated with mitogen-activated protein kinases (MAPK); they may also induce calcium signalling [3,29].

3. Plant prenyllipid antioxidants: structure, occurrence, biosynthesis, and function

3.1. Isoprenoid chromanols and quinones

Important plant prenyllipid antioxidants belonging to isoprenoid chromanols and quinones are shown in Figure 5, and those commonly occurring in chloroplasts are marked with a dark green colour. All of these compounds have amphipathic properties, because their molecules are composed of a polar head-group (a chromanol or quinone ring) and an apolar prenyl side-chain [31,32]. The main chromanol present in plant green tissues is α-tocopherol (α-Toc) [33]. The other forms of tocopherols (called β-, γ- and δ-vitamers) all contain phytol-derived saturated side-chain, but differ in the number and position of methyl substituents in the chromanol ring. Among them, γ-tocopherol (γ-Toc) is found in leaves in lesser amounts than α-Toc, however, it is often abundant in seed oils of many plant species [34]. The other vitamers can also be found in plant seeds, together with α-, β-, γ-, δ-tocotrienols (T₃s), which contain geranylgeranyl-derived side-chain with three unsaturated bonds. The occurrence of certain Tocs and T₃s in seeds and their relative abundance depend on the plant species [32]. In some species γ- and δ-Toc were also found in cuticular waxes [33]. Plastochromanol-8, which contains head-group same as γ-Toc and much longer, unsaturated side-chain is present in green tissues in minor amounts [35]. The other chromanols, such as tokomonoenols (T₁s) and tokodienols, containing one and two unsaturated bonds in their side-chains, respectively, as well as other derivatives, including demethylated or phosphorylated forms, have recently been discovered. They occur in small amounts in some plant species [4].

Among isoprenoid benzoquinones, plastoquinone-9 (PQ-9), together with its reduced form, plastoquinol-9 (PQH₂-9) that has hydroxyl instead of ketone groups in the ring, are present in chloroplasts in high amounts [31]. Its analogues containing shorter side-chains, which may also differ with the degree of saturation, as well as forms varying in the number of methyl groups in the quinone ring (i.e. 6-methyl-dihydrophytylplastoquinone, 3-demethylplastoquinone-9), have also been described in the literature [4,36]. Ubiquinone (UQ) and ubiquinol (UQH₂) occur in the inner membrane of mitochondria [37]. The most common number of isoprenoid residues in their side-chains is 9 and 10, however, forms with shorter chains have also been found in minor amounts. The exact composition of UQs depends on the plant species [38,39]. Taking into account naphthoquinones, photosynthetic eukaryotes typically contain phylloquinone (PhQ), but in some algal species menaquinone-4, containing the side-chain derived from geranylgeranyl, instead of the phytol-derived one was found. The PhQ derivative that contains the -OH group in the side chain has also been described in the literature [40,41,42].

The oxidized derivatives of α-Toc, PC-8 and PQ-9, which are α-tocopheryloquinone (α-TQ), hydroxy-plastochromanol (PC-OH), and plastoquinone C (PQ C), respectively, were drawn in Figure 5 using a sap-green colour. PQ derivatives that contain more -OH groups in the side-chain have also been described in the literature [43]. All these compounds occur in plants in small amounts, but their level is known to increase under stress conditions [43,44,45].

Due to their crucial role in photosynthesis, PQ and PhQ are present in both cyanobacteria and photosynthetic eukaryotes. In the latter organisms, PQ occurs in chloroplasts: in thylakoid membranes, plastoglobules, which are lipid storage sites, as well as in the inner chloroplast envelope, where it is synthesised [4]. PhQ is a cofactor in PS I, but minor amounts of this compound were found in plastoglobules, chloroplast envelope, and cell membranes [31]. The occurrence of both PQ and PhQ is not limited to green tissues, as these prenyllipids are also present in small amounts in non-photosynthetic organs, such as flowers, fruits, roots and bulbs [31]. UQ is commonly present in eukaryotes and plays a key role in respiration. In mitochondria, it occurs in the inner membranes [31,37].

Isoprenoid chromanols are synthesised by most cyanobacteria. These compounds are also ubiquitous in photosynthetic eukaryotes, but their amounts vary depending on the taxonomic affiliation. In particular, higher plants are known to contain high levels of isoprenoid chromanols, which are also more structurally diversified [4,32]. In chloroplasts, isoprenoid chromanols occur in thylakoids, envelopes, and plastoglobules. Non-green plastids also contain chromanols. In particular, these prenyllipids can be stored in leukoplasts in seed cells. However, in addition to plastids, isoprenoid chromanols in seeds can also be found in oleosomes, which are lipid droplets surrounded by a protein-lipid monolayer and dispersed in the cytoplasm. These lipid bodies can store up to 40% of the seed chromanol pool [4,46,47]. The presence of chromanols in other cell compartments was reported in the literature, but the purity of the fractions used for the analyses has been questioned [33]. Concerning plant organs, the highest levels of isoprenoid chromanols occur in green tissues and seeds, in particular those accumulating lipids as the major storage material. These compounds can also be found in non-photosynthetic plant organs, but generally their content is lower compared to leaves [4]. Chromanol levels usually increase during acclimatisation to environmental stresses [48].

In the biosynthetic pathway of isoprenoid quinones and chromanols, the side-chain and head-group precursors are synthesised separately [4]. Subsequently, specific prenyltransferases catalyse their condensation, and the resulting compound is further modified. In this paper, the synthesis of Tocs, PQ and PC-8 is described in more detail, as these compounds are well known as efficient antioxidants present in chloroplasts.

The Toc biosynthesis pathway is shown in Figure 6. Homogentisate (HGA), which serves as a precursor of the head-group of isoprenoid chromanols, is synthesised from p-hydroxyphenylpyruvate, a compound derived from tyrosine [49]. The synthesis of HGA is catalysed by p-hydroxyphenylpyruvate dioxygenase (HPPD, encoded by the PDS1 gene) that is present in chloroplasts or cytosol, depending on the species [50]. The side-chain is synthesised via multistep condensation of five-carbon precursors, isopentenyl pyrophosphate (IPP), and dimethylallyl pyrophosphate (DMAPP). There are two pathways of IPP and DMAPP synthesis, that is, 1-deoxy-D-xylulose-5-phosphate (DXP) pathway, also called the methylerythritol phosphate pathway, and the mevalonate (MVA) pathway [51]. Cyanobacteria possess the DXP pathway, whereas higher plants have both, DXP in plastids and MVA in the cytosol [49]. Considering eukaryotic algae, most of them also contain both pathways, but there are eukaryotic algae that have lost one of the pathways [52,53]. It is thought that in plants the IPP and DMAPP used for the synthesis of plastid isoprenoids are mainly derived from the DXP pathway. However, it was shown that in the situation of overproduction of these precursors in the cytoplasm, they can be transported to chloroplasts [50]. Isoprenoid chains of different lengths are synthesised through condensation of IPP and DMAPP by various polyprenyl pyrophosphate synthases. Among them, geranylgeranyl pyrophosphate synthase (GGPPS) catalyses the subsequent head-to-tail condensation of four isoprenoid units. The resulting geranylgeranyl pyrophosphate (GGPP) is a common precursor for the biosynthesis of various isoprenoids produced in plastids [49,50]. In the Toc biosynthetic pathway, GGPP is then reduced to phytol pyrophosphate (PPP). Higher plants are also known to utilise phytol derived from degradation of Chls. In fact, this route of formation of Toc side-chain is thought to dominate in these organisms. Specific kinases, encoded by the VTE5 and VTE6 genes, catalyse subsequent phosphorylations of phytol to PPP [34,50]. Homogentisate phytyltransferase (HPT, encoded by the VTE2 gene) carries out the condensation of HGA with PPP. The product of this reaction can then undergo cyclisation by tocopherol cyclase (TC, encoded by the VTE1 gene), leading to the formation of δ-Toc, or methylation by 2-methyl-6-phytyl-1,4-benzoquinone methyltransferase (MPBQ MT, encoded by VTE3). Subsequent cyclisation of the methylated intermediate by TC results in γ-Toc formation. Both δ-Toc and γ-Toc are substrates for γ-tocopherol methyltransferase (γ-TMT, encoded by VTE4), and the products of these reactions are β-Toc and α-Toc, respectively [51]. HPT can also catalyse condensation of HGA with tetrahydrogeranylgeranyl pyrophosphate in the biosynthetic pathway of T₁s [54]. In the synthesis of T₃s, HGA is condensed with GGPP by homogentisate geranylgeranyl transferase and the subsequent reactions are catalysed by TC, MPBQ MT, and γ-TMT, like in the Toc and T₁ biosynthetic pathways [51]. The last steps of synthesis of isoprenoid chromanols occur mostly in the chloroplast envelope, where HPT, MPBQ MT and γ-TMT localise. On the other hand, TC was found in plastoglobules [55].

The synthesis of PQ-9 and PC-8 is shown in Figure 7. In higher plants, HGA serves as a head-group precursor for PQ. On the other hand, cyanobacteria, which use HGA for the synthesis of Tocs similar to plants, for the synthesis of PQ use p-hydroxybenzoate, which is also a precursor of head-group in the biosynthesis of UQ in eukaryotes and those of proteobacteria that contain UQ [56,57]. The side-chain precursor, solanesyl pyrophosphate, is synthesised by the subsequent addition of five IPP to GGPP, carried out by solanesyl pyrophosphate synthase (SPS). In higher plants, homogentisate solanesyl transferase (HST) is an enzyme responsible for the condensation of SPS with HGA. The product of this reaction is then methylated by MPBQ MT, leading to the formation of PQH₂-9. Enzymatic or non-enzymatic oxidation of PQH₂-9 is a source of PQ-9. Some PQH₂-9 may undergo cyclisation catalysed by TC, resulting in PC-8 formation [4]. The synthesis of PQ in cyanobacteria requires different prenyltransferase, which gene is homologue to the one participating in the synthesis of UQ, and different steps of further modifications of the head-group [56].

In the synthesis of PhQ head-group precursor is derived from chorismate, while PPP serves as the precursor of the side-chain. Most enzymes that participate in PhQ biosynthesis are located in plastids, but some steps take place in peroxisomes [58,59,60]. The synthesis of UQ occurs in mitochondria and its detailed description can be found in the review by Xu et al. [39].

Due to their amphipathic properties, isoprenoid chromanols and quinones localise in membranes and lipid storage sites, where they display their protective properties. Isoprenoid chromanols are well-known potent lipophilic antioxidants. Being the most common vitamer, α-Toc has been studied most extensively in this respect, both in vitro and in vivo. This compound was shown to be an efficient scavenger of radicals, including O₂^∙−/ HO₂^∙ and those formed during radical-mediated lipid peroxidation: R^∙, RO^∙, and ROO^∙ [61,62]. α-Toc is also known to efficiently quench and scavenge ¹O₂ [63,64]. The antioxidant properties of other Toc vitamers, T₃s and PC-8, have also been examined in various systems [63,64,65,66,67].

In radical scavenging reactions, isoprenoid chromanols donate a hydrogen atom from the hydroxyl group in their chromanol ring. Hydrogen-donating power depends on a ring structure, and decreases in order α > β > γ > δ [61]. In some assays this order corresponded well to the antioxidant efficiency of certain vitamers [66,67], in other cases the order of decreasing antioxidant potency was α > β ≈ γ > δ [65,66]. There were also cases in which α-Toc did not exceed other vitamers in its antioxidant action [66]. The chromanoxyl radical, formed as a result of radical scavenging by isoprenoid chromanols, is relatively stable because of delocalisation of electrons in the aromatic ring. It can be re-reduced to the corresponding Toc by ascorbate (Asc), reduced isoprenoid quinols such as PQH₂-9, and phenolic compounds [64,68,69,70]. However, if the reductant is scarce or absent and when peroxidation occurs in dispersed systems, such as liposomes or low density lipoproteins (LDLs), tocopheroxyl radical lifetime can be prolonged, which favours hydrogen abstraction from other molecules and thus, chain-carrying pro-oxidant action [64,71,72,73]. Fortunately, chloroplasts are the site where Asc and PQH₂-9 are usually abundant [4,74]. Considering the impact of the side-chain on the antioxidant action, α-T₃ turned out to be more effective than α-Toc in counteracting radical-mediated peroxidation in liposomes [66,75]. Serbinova et al. postulated that this effect resulted from a higher recycling efficiency of α-T₃ from chromanoxyl radicals, more uniform distribution of α-T₃ in membrane bilayer, and a greater disordering of membrane lipids by α-T₃, which makes interaction of chromanols with lipid radicals more efficient [75].

Taking into account the quenching of ¹O₂, the rate constants of reaction of α-Toc with ¹O₂ in organic solvents were higher than those of other vitamers and PC-8, while the rates of the T₃s vitamers were comparable to their respective Tocs. However, for all compounds, the order of magnitude of the rate constants was the same [63]. Tocs ¹O₂ scavenging rates are lower than their ¹O₂ quenching rates for approximately two orders of magnitude and were shown to decrease in order α > γ > δ > β [76]. Scavenging of ¹O₂ by α-Toc results in the formation of 8a-hydroperoxy-α-tocopherone, which is unstable and may be further converted to α-TQ or re-reduced to α-Toc by Asc [55]. In chloroplasts α-TQ can be reduced to quinol form (α-TQH₂) [77], which displays antioxidant properties similar to those of other isoprenoid quinols (see below) and can also be enzymatically recycled back to α-Toc [78]. The reactivity of PC-8 towards radicals and ¹O₂ is similar to that displayed by γ-Toc. However, due to its longer side-chain, PC-8 was suggested to be a particularly effective antioxidant in the hydrophobic core of the lipid bilayer [64].

While α-Toc has a fully substituted benzene ring in the chromanol head-group, γ-Toc has an unsubstituted 5-position and therefore is able to trap electrophiles, including reactive nitrogen species (RNS) [79,80].

In addition to their antioxidant properties, isoprenoid chromanols were also postulated to participate in signalling, mostly through interference with ROS and RNS-mediated pathways [8,81]. For example, the phytohormone jasmonic acid (JA) belongs to oxylipins. Because lipid hydroperoxide is an intermediate in its synthesis [82], it was postulated that by inhibition of lipid peroxidation α-Toc is able to limit JA synthesis [83]. Experiments in corn mutant sxd1 lacking α-Toc showed the importance of this prenyllipid for proper formation of plasmodesmata and phloem loading [84,85,86]. Isoprenoid chromanols have also an impact on membrane properties. α-Toc was shown to increase membrane rigidity and ordering, decrease ion leakage, and favour the formation of reversed hexagonal structures [33,87,88].

Isoprenoid quinones are the most recognised as electron carriers in electron transfer chains, such as photosynthetic, respiratory, and chlororespiratory ones. These compounds also function as enzyme cofactors and participate in cellular signalling [31,36,37]. Their reduced forms are well-known for their potent antioxidant properties [4]. Quinones may also act as antioxidants, but their activity is lower compared to that of quinols. Isoprenoid quinols can scavenge inorganic (HO₂^∙, perferryl radical) and lipid radicals, as well as quench and scavenge ¹O₂ [63,64,89,90,91]. PQH₂ was also shown to scavenge H₂O₂ in organic solvents [92]. The scavenging of radicals by isoprenoid quinols results in the formation of semiquinone radicals, which undergo disproportionation to quinones and quinols [90]. Quinones are known to react with O₂^∙− which leads to the formation of semiquinone radical [93,94]. However, quinols and semiquinone radicals can also act as pro-oxidants [90].

The rate constants of ¹O₂ quenching by PQH₂-9 and UQH₂-10 in organic solvents were lower than those of α-Toc, but similar to values measured for other Toc vitamers. PQ-9 was also shown to quench ¹O₂, however, the rate constant of this reaction was a few times lower compared to that of PQH₂-9 [63]. Taking into account the scavenging of ¹O₂ by PQ/PQH₂, apart from the head group, unsaturated bonds in the side-chain can also be the target of oxidation, leading to the formation of hydroxy-derivatives. These products are considered markers of enhanced ¹O₂ formation in vivo, together with PC-OH [43,44]. An in vivo study on C. reinhardtii exposed to high-light stress showed that PQH₂ is the main prenyllipid responsible for the detoxication of ¹O₂ in chloroplasts [91]. Similarly, transgenic A. thaliana containing increased amounts of PQH₂ and PC-8 due to the over-expression of the sps1 gene was more resistant to high light than wild type, while such an effect was not observed in the line with increased levels of α- and γ-Toc [95]. The amount of PQ/PQH₂ increases under stress conditions known to cause oxidative stress in plants [36]. The antioxidant function of UQ/UQH₂ in plants has not been examined as widely as that of Toc and PQ/PQH₂, but there are some literature data concerning this topic [37].

PQ is known to play a role in chloroplast-derived signalling. PQ pool is thought to act as a redox sensor responsible for the initiation of response to changing light conditions. In experiments carried out in plants and algae using inhibitors of photosynthetic electron transfer or selective excitation of PS I and II it was shown that the change in the PQH₂/PQ ratio had an impact on the expression of important genes, both chloroplast and nuclear, such as those encoding PS II core peptide D₁, PS I core peptides PsaA and PsaB, LHC II, ascorbate peroxidases, superoxide dismutase and nitrate reductase [96,97,98,99,100,101,102,103,104,105]. Similarly to Tocs, due to its antioxidant properties, PQ/PQH₂ was postulated to participate in cellular signalling through interaction with ROS-mediated signalling pathways [36].

The redox state of the PQ pool is also known to regulate the activity of the kinase STN7 and its homologues, responsible for the induction of the state transition of the photosynthetic apparatus from state 1 to state 2. This process involves dissociation of LHC II antennae from PS II and their association with PS I, as well as aggregation of LHC II in thylakoid membranes. State transitions play a role in balancing the energy supply to photosystems and the regulation of cyclic electron transfer around PS I [106,107].

Furthermore, PQ is a cofactor of enzymes necessary for carotenoid biosynthesis in plants, algae, and cyanobacteria, i.e. phytoene desaturase and ζ-carotene desaturase [108].

3.2. Carotenoids

Carotenoids are a large class of natural pigments, ancient in their evolutionary origin, and relatively common among living organisms [109,110]. So far, more than 1100 natural compounds belonging to this group have been discovered [111]. Being accessory photosynthetic pigments, crucial for light harvesting and photoprotection, carotenoids are synthesised by all photosynthetic organisms, both prokaryotic and eukaryotic [9]. They are also produced by some non-photosynthetic bacteria, archaebacteria, protozoa, and fungi [112]. Animals need to obtain these pigments in food; however, there is a known aphid species that can produce carotenoids on its own [113].

Carotenoids are tetraterpenoids, which means that they are synthesised by condensation of eight isoprenoid units. However, there are known exceptions, that is, heliobacteria produce carotenoids made of six isoprenoid units as well [114]. The typical C₄₀ backbone can also be further extended, producing C₄₅ or C₅₀ carotenoids, while shortening of the molecules leads to the formation of apocarotenoids [109]. These pigments are divided into two subgroups, that is, carotenes, which are hydrocarbons, and xanthophylls, which contain one or more oxygen atoms [9]. All carotenoids are long molecules, containing multiple double bonds, owning to which they efficiently absorb visible light, in the range 400-550 nm [112]. Taking into account the conformation of double bonds, all-trans carotenoids are more prevalent in nature; however, cis-isomers may be formed enzymatically or as a result of exposure to heat or light [111,115]. Some of these compounds are acyclic, whereas the rest contain ring(s), usually at their ends. They differ in degree of saturation, number of conjugated double bonds, and presence and distribution of various substituents [111,116]. Due to the diversity of substituents, there are known xanthophylls that are alcohols and their esters, aldehydes, ketones, acids and their esters, epoxides, ethers, and glycosides [9].

The detailed carotenoid composition of a given organism depends on the systematic affiliation; therefore, these compounds are valuable chemotaxonomic biomarkers [110,116,117]. Higher plants are known to synthesise a narrower array of these pigments, compared to photosynthetic bacteria and algae [116,118]. Carotenoids commonly occurring in plant green tissues are shown in Figure 8, with those present in higher amounts drawn in orange. Among them, lutein is the most abundant [119,120].

In addition to these compounds, the intermediates of their biosynthetic pathway, such as lycopene, δ- and γ-carotene, as well as some derivatives, such as epoxy-lutein, can also be found in small amounts and in non-green tissues [9,119,121]. The most abundant carotenoid of cyanobacteria is usually β-carotene, but they synthesize its hydroxy-derivatives zeaxanthin and nostoxanthin, as well as ketocarotenoids, such as canthaxanthin and echinenone, and carotenoid glycosides, for example myxoxanthophyll [122]. In eukaryotic algae, the diversity of carotenoids is remarkable [116]. The carotenoid composition of green algae is similar to that of higher plants; however, they also synthesise other compounds, such as astaxanthin, siphonaxanthin, or loroxanthin. The dominant carotenoid in unicellular red algae is zeaxanthin, whereas macrophytic red algae contain lutein as their main carotenoid. The major carotenoids of stramenopiles are diadinoxanthin and fucoxanthin. These compounds can be also found in haptophytes; however, diadinoxanthin is not as widespread as in the group mentioned above. Cryptophytes contain α-carotene and acetylene xanthophylls, mainly alloxanthin. In dinoflagellates, peridinin is a characteristic xanthophyll. Euglenids contain carotenoids typical of green algae, but also those present in stramenopiles, such as diadinoxanthin [117,123,124].

In cyanobacteria, carotenoids are present in thylakoids, but can also be found in the cell membrane and cell wall [109]. In photosynthetic eukaryotes, these pigments occur in chloroplasts, where they are bound to photosystems and antennae. However, some fraction diffuses freely in membranes, mainly thylakoid ones, and may be stored in plastoglobules. In non-green tissues, carotenoids are present in large amounts in chromoplasts and in minor amounts in leucoplasts [110]. In some algae, carotenoids also occur in the eyespot and oil droplets [25,125]. Considering organ distribution in higher plants, these pigments are present in all green tissues, but can also occur in certain flowers, fruits, roots, and bulbs [110,121]. Carotenoids can also be found in seeds and pollen [119].

Carotenoid biosynthesis starts with the condensation of IPP and DMAPP, resulting in the formation of GGPP. The synthesis of these precursors has been briefly described in Subchapter 3.1. Here, it is only worth mentioning that in higher plants carotenoids are synthesised in plastids; therefore, C₅ precursors are derived from the DXP pathway [110]. Phytoene synthase catalyses the head-to-head 1-1 condensation of two GGPP molecules, resulting in the formation of 15-cis phytoene [112]. To obtain lycopene, which is a common precursor for the synthesis of many C₄₀ carotenoids, 15-cis phytoene must undergo desaturation and isomerisation. In the so-called all-trans pathway, present in carotenoid-synthesising fungi, archaebacteria, and bacteria other than cyanobacteria, these reactions are catalysed by a single enzyme, called CrtI [108,126]. Cyanobacteria (with the known exception of Gloeobacter violaceus using the all-trans pathway) have another more complicated poly-cis pathway, and because plastids are of cyanobacterial origin, photosynthetic eukaryotes also use it [127]. This pathway involves four enzymes, two desaturases, and two isomerases (Figure 9). First, phytoene desaturase (PDS) in two steps introduces two double bonds in cis-conformation at 9 and 9’ positions, forming 9-15-9’-tri-cis-ζ-carotene and using PQ as electron acceptor. Next, 15-cis-ζ-carotene isomerase (Z-ISO) catalyses isomerisation at the 15 position. This reaction may also occur non-enzymatically, under high light. Resulting 9-9’-di-cis-ζ-carotene is utilized by ζ-carotene desaturase (ZDS) which in two steps introduces two double bonds in cis-conformation at the positions 7 and 7’, leading to the formation of 7,9,7’,9’-tetra-cis-lycopene. Similarly to PDS, ZDS uses PQ as a co-substrate. Finally, carotenoid isomerase (CRTISO) performs two steps of isomerisation, producing all-trans lycopene. Similarly to isomerisation at the earlier stage of lycopene synthesis, these reactions can also occur non-enzymatically in high light [108,112].

In plants, lycopene can be found in high amounts, for example in tomato and watermelon fruits, but more often this compound is further modified to obtain cyclic carotenoids [110]. Two cyclases, lycopene β-cyclase (LCYβ) and lycopene ε-cyclase (LCYε), are responsible for the formation of β- and ε-ionone rings at the end of the molecule, respectively. At this stage, the carotenogenic pathway of plants undergoes bifurcation (Figure 9). Both δ-carotene, containing ε-ionone ring, and γ-carotene, containing β-ionone ring, can be further cyclised by LCYβ, resulting in α- and β-carotene, respectively [112,128]. Second cyclisation of δ-carotene catalysed by LCYε, leading to the formation of ε-carotene is very rare in plants [127]. Other cyclisation reactions are also possible, but not common; for example, plants belonging to the pepper family are known to produce carotenoids with cyclopentane ring(s) [110]. Most cyanobacteria do not contain LCYε and therefore have only one branch of the pathway [122]. In eukaryotic algae, some clades have both branches, whereas the other have only LCYβ [128].

Further modifications of carotenes, carried out by various enzymes, mainly hydroxylases and epoxidase, result in the formation xanthophylls (Figure 10). Hydroxylases are specific for the ring, into which they introduce hydroxyl group, therefore they were called β- and ε- carotenoid hydroxylases (CHYβ and CHYε, respectively). Multiple genes of carotenoid hydroxylases were found in Arabidopsis and tomato [110]. Hydroxylation of both rings of α-carotene is necessary to produce lutein, whereas hydroxylation of both rings of β-carotene results in zeaxanthin (Zx) formation. The two-step epoxidation of Zx by zeaxanthin epoxidase (ZE) leads to the synthesis of violaxanthin (Vx). The reverse reaction, i.e. the two-step de-epoxidation of Vx to Zx, catalysed by violaxanthin de-epoxidase (VDE), is important for photoprotection and will be discussed later. Vx can also be converted to neoxanthin (Nx) by neoxanthin synthase (NSY) [121].

Being very efficient in light energy capture and absorbing light in a range not absorbed by Chls and bacteriochlorophylls (BChls), carotenoids are accessory photosynthetic pigments present in photosystems and the majority of antennae types [129]. Furthermore, these compounds are usually necessary for proper assembly and stabilisation of pigment-protein complexes of the photosynthetic apparatus [124,128,130].

In addition to their role in light-harvesting, carotenoids are crucial for photoprotection, because of their ability of fast and efficient deactivation of their excited states via thermal dissipation. Rapid quenching of triplet excited states of (B)Chls allows carotenoids to prevent the formation of ¹O₂ in the photosynthetic apparatus [9,129,131]. In addition, the dissipation of excess energy absorbed by antennae and photosystems diminishes the risk of O₂^∙− formation due to over-reduction of the photosynthetic electron transfer chain [15,132].

The crucial photoprotective mechanism of higher plants, called qE (Zx-dependent ΔpH-induced nonphotochemical quenching of chlorophyll fluorescence), requires a proton gradient across the thylakoid membrane and de-epoxidised xanthophyll Zx to function. The latter is formed in a violaxanthin cycle, i.e. interconversion of Vx, antheraxanthin (Ax) and Zx, depending on light conditions. Two opposite reactions of the cycle are catalysed by enzymes belonging to the lipocalin superfamily: Zx epoxidase (ZE) and Vx de-epoxidase (VDE) [133]. In non-stress conditions, the level of Zx in plant leaves is low, while the level of Vx is high. Under high light VDE, activated by low pH in the thylakoid lumen, removes epoxy-groups and introduces double bonds in both rings of Vx, producing Zx (Figure 10). In low light or darkness, ZE catalyses the reverse reaction of epoxidation of Zx to Vx [134]. The presence of Zx influences the interactions between PS II antennae and the PsbS protein crucial for qE function in higher plants [121]. In addition to qE, Zx is also known to participate in the photoprotective mechanism called qZ, which does not require a pH gradient across the thylakoid membrane for functioning. This mechanism was found to occur in plants and green algae [135,136].

The violaxanthin cycle is the most common cycle that involves photoprotection-related interconversion of xanthophylls. However, five other cycles have been discovered in certain groups of algae and some plant species, two of them being the incomplete violaxanthin cycle, while the rest are based on other xanthophylls. Among the latter, the most important is the diadinoxanthin cycle, in which diadinoxanthin is de-epoxidised to diatoxanthin under high light [137]. This cycle was found to occur in many clades of algae, including diatoms, brown algae, dinophytes, and haptophytes [138]. Interestingly, the contribution of the xanthophyll cycle to photoprotection in green algae depends on species and growth conditions and is rather low compared to land plants [139,140]. In these organisms, qE depends on the presence of LHCSR proteins, in which carotenoids, most probably lutein, are responsible for the dissipation of excitation energy [141]. LHCSR proteins were also found in mosses [142].

In cyanobacteria, the keto-carotenoid molecule is bound to Orange Carotenoid Protein (OCP), which participates in the regulation of energy dissipation under high light. The absorption of blue-green light causes the isomerisation of carotenoid and subsequent change in OCP conformation. The active, red form of OCP binds to the phycobilisome and facilitates the dissipation of excess energy captured. In low light or darkness, the red form is converted to an inactive orange form [143].

The mechanisms of thermal dissipation of excessive energy in other clades of algae require further study. Additionally, carotenoids can also act as sunscreens, reducing the penetration of light into the chloroplast or whole cell [144]. Being present in algal eyespots, carotenoids are also important for proper light perception and phototaxis [125].

Carotenoids not only prevent ROS formation, but are also potent lipophilic antioxidants, present both in pigment-protein complexes and freely diffusing in membranes [9]. These compounds are known to efficiently quench ¹O₂. The principal quenching mechanism is Dexter-type energy transfer, followed by dissipation of excitation energy as heat by skeletal vibration of the carotenoid molecule. Efficient energy release occurs in carotenoids having at least nine conjugated double bonds, and the higher the number of these bonds, the better is certain compound in quenching [145]. Therefore, lycopene is one of the most efficient antioxidants in this respect. The quenching of ¹O₂ by many natural carotenoids occurs with rate constants approaching diffusion control [76]. On the other hand, the formation of carotenoid aggregates was suggested to result in a decrease in quenching efficiency [146]. The rate constants for ¹O₂ quenching depend not only on the compound tested, but also on the model system used; however, in many experimental setups the values determined for carotenoids exceeded those of α-Toc [147]. The scavenging of ¹O₂ by carotenoids is also possible, but it is a minor side reaction compared to quenching, as it is estimated to be responsible for 0.05% of total ¹O₂ detoxifying activity by these compounds [76,145]. This process leads to the decomposition of carotenoids, and some products of their oxidation are known to be involved in stress signalling [148]. Carotenoids may also scavenge radicals, including O₂^∙−/ HO₂^∙ and lipid radicals R^∙, RO^∙, and ROO^∙ [149,150,151]. Therefore, these compounds can inhibit lipid peroxidation. However, carotenoids act as chain-breaking antioxidants under low O₂ concentrations, whereas at higher ones these pigments act as chain-carrying pro-oxidants. The pro-oxidant effect is due to the reaction of carotenoid radicals with O₂, which leads to the formation of Car-OO^• able to abstract hydrogen from the lipid molecule [149].

Carotenoids that freely diffuse in membranes have an impact on membrane structure and microviscosity. Therefore, they modulate the molecular dynamics of the lipid bilayers. The impact of certain compound on membrane properties depends on its chemical structure [112,128]. Carotenes, which are non-polar molecules, are thought to be entirely embedded in the hydrophobic core of the membrane. On the other hand, xanthophylls, containing polar group(s), usually at their ends, tend to localise so that these groups are anchored in the polar zone(s) of the lipid bilayer. Xanthophylls having all-trans conformation and polar groups at both ends are thought to be localised so that their terminal groups are anchored in the two opposite polar zones of the bilayer. Such a position enables the formation of hydrogen bonds in the polar zones and van der Waals interactions in the hydrophobic core. Both of these interactions modify membrane stability and rigidity, and have an impact on the efficiency of diffusion through the membrane [128]. Concerning their impact on membrane fluidity, carotenoids can be divided into (1) non-affecting xanthophylls, having polar group only at one end, (2) fluidizing carotenes, and (3) rigidifying xanthophylls, containing polar groups at both ends. Photosynthetic organisms take advantage of these properties of carotenoids, changing their composition to maintain the desired membrane fluidity under changing environmental conditions [128].

Cleavage and further modification of carotenoids leads to the formation of apocarotenoids, which participate in signalling or act as volatile aromatic compounds [112,127]. The most widely known example of such compounds is the phytohormone abscisic acid (ABA), which plays a crucial role in plant acclimatisation to drought and other abiotic stresses, and in the regulation of dormancy. ABA is also involved in the regulation of plant development and senescence [152]. Another example of carotenoid-derived plant hormones are strigolactones, which play a role in symbiotic interactions with arbuscular mycorrhizal fungi, regulate shoot and root growth, and leaf senescence [153]. Apocarotenoids other than ABA and strigolactones have also been postulated to participate in the regulation of plant growth, development, and stress response [110,148,154].

4. Cold stress in plants, its effects and protective mechanisms

Temperature conditions on Earth vary in a wide range, therefore living organisms differ with their tolerance to that environmental factor. On the basis of displayed thermal optimum for active metabolism they were classified into psychrophiles, mesophiles, thermophiles and hyperthermophiles. According to Goldstein [155], psychrophilic organisms have a growth maximum below 20 °C, mesophilic in the range 20 °C–45 °C, thermophilic in the range 45 °C–80 °C, and hyperthermophilic above 80 °C. However, the values given for certain subclass vary depending on the literature source (for comparison, see [156,157,158]). Photosynthetic eukaryotes are mainly psychrophiles and mesophiles, some thermophilic species can also be found [156,159]. Plant tolerance to temperature depends on the strategies evolved to adapt to climate conditions. Species that occur in tropical and subtropical areas lack the capacity to acclimate to low temperature and, therefore, are sensitive to cold stress. Plants growing in temperate climate can tolerate seasonal changes in temperature, while those living in subarctic and arctic zones can withstand severe frosts [2,160]. Temperature tolerance in dormant stages (seeds, spores) and organs is greater than that of metabolically active stages and tissues [158]. In actively metabolising plants, tolerance varies depending on organ, tissue, and age – generative organs, meristems, and seedlings are usually more sensitive to temperature stress [161,162]. When considering plant reproduction, especially pollen is vulnerable to chilling, which causes its sterility, inhibits pollen germination, and slows down the elongation of the pollen tube. Cold stress may also lead to abnormal ovule development [163,164]. The damage caused by low temperature depends on the length of cold period and the rate of cooling and rewarming. The sudden decrease in temperature is more damaging than the slow one [158,161]. The co-existence of other stress factors is also an important feature [156,158,165].

Low temperature stress can be subdivided into chilling stress and freezing stress, the latter occurring when the temperature drops below 0 °C causing the threat of the formation of ice crystals within the organism [2,160]. Chilling-sensitive plants, such as corn, are damaged at temperatures between 10 and 15 °C. They were subdivided into those having all organs sensitive to low temperature and those in the case of which only some organs, for example buds and flowers, are cold-sensitive [158]. Considering freezing stress, plants were subdivided into freezing-sensitive plants, which cannot withstand conditions in which water crystallisation occurs, and freezing-tolerant plants, able to cope with such conditions. In plants living in climate zones where frosts occur in winter, tolerance to freezing stress changes throughout the year, being developed in the autumn and lost in the spring [158,161].

Temperature has an impact on many crucial physicochemical parameters, including diffusion rates of metabolites and gases, solubility of compounds, rate of chemical reactions, viscosity of aqueous solutions, and strength of molecular interactions, the latter related to maintaining proper structure of biomolecules and membrane fluidity [158,166,167]. Proper membrane fluidity is a crucial factor for efficient functioning of electron transfer chains and other processes involving membrane-bound enzymes, membrane transport proteins, and membrane-bound receptors. In low temperature, the lipid bilayer can also undergo a transition from the liquid-crystalline phase to the rigid gel phase. The lipid phase transition, apart from its effects on lateral diffusion and structural changes in membrane-embedded proteins, causes membrane leakage, resulting in disturbance of ion homeostasis, ATP synthesis, and signalling [158,161,167,168]. Due to the reasons listed above, the decrease in temperature leads to the decrease in the general rate of metabolism and changes in cell structure [158,167].

As was mentioned in the Introduction, under low temperature photosynthetic apparatus is especially prone to damage, because under such conditions the efficiency of using absorbed light energy for desired photochemical reactions is lower, and therefore the threshold of light intensity above which light is excessive is decreased when compared to optimal temperature. The extra energy absorbed by photosynthetic apparatus fuels ROS formation, leading to the appearance of oxidative stress [4,162,169,170]. The increase in oxidative stress markers in cold-exposed plants is a phenomenon well-documented in the literature (Table 1). Another adverse effect of low temperature is the alteration of gene expression due to the formation of double-stranded RNA structures [171]. Cold stress disrupts water homeostasis. The malfunction of stomata regulation, loss of turgor, and inhibition of elongation growth are observed in plants exposed to low temperature. Under these conditions, water uptake is less efficient due to an increase in viscosity of aqueous solutions and a decrease in membrane fluidity [158,164,172,173]. The uptake of nutrients by roots, as well as xylem and phloem transport, are also slowed down at low temperatures [164,174,175]. Lower efficiency of phloem loading and transport adds to the decrease in photosynthesis rate, due to the negative feedback regulatory loop [176]. The macroscopic effects of cold stress observed in plants are growth inhibition, yield reduction, chloroses, necroses, wilting, curling, crinkling and browning of leaves [167,173,177].

At temperatures below 0 °C, water begins to crystallise in plant tissues. Because dissolved substances decrease the temperature of phase transition of the solution into solid state, the cytoplasm with its high concentrations of metabolites and ions needs lower temperature to freeze than the extracellular aqueous solutions [2,177]. Usually, ice formation starts in peripheral vascular bundles and intercellular spaces in aboveground organs. After the initiation of crystallisation in the bundles, the ice spreads rapidly in the xylem [158,193]. The formation of extracellular ice results in water efflux from the cells. Under such conditions, dessication and ion stress occur, with all their adverse effects on metabolism and cell structure [194,195,196]. As the water expands during freezing, mechanical strain is exerted on the plant tissues. Also, freezing and thawing of xylem sap leads to xylem cavitation and a drastic decrease in the efficiency of water transport [194,195]. In severe frosts, intracellular ice formation occurs, which is deadly to the cell, as growing crystals damage organelles [161]. Another adverse condition faced by plants during winter is physiological drought due to the freezing of water in the soil [197]. Additionally, snow cover causes light deprivation and limits oxygen availability in the soil. It can also mechanically damage plants [198,199].

The sensing of cold and the following signal transduction pathways have been the subject of intensive studies [164,173,200]. Their detailed description is beyond the scope of this review. Briefly, it is thought that the decrease in temperature is perceived at the membrane and cytoskeleton levels. The changes in pools of certain metabolites can also be an indicator of low temperature. Secondary messengers involved in cold stress signalling include Ca²⁺, ROS, and NO. Taking into account proteins, important roles are played by COLD-RESPONSIVE PROTEIN KINASE 1 (CRPK1), calcium/calmodulin-regulated kinases and MAPK cascade [200]. Phytohormones are other signalling compounds that play a role in response and acclimatisation to low temperature stress [173]. Photoreception is also related to cold sensing and acclimatisation, and the receptors involved are phytochromes and cryptochromes [173,200].

The mechanisms of acclimatisation and adaptation to low temperature are shown in Table 2. Relevant to this review are those related to membrane fluidity maintenance, antioxidant response, and photoprotection. The increase in the number of unsaturated double bonds in membrane fatty acids is an important mechanism of cold acclimatisation. However, PUFA are more prone to peroxidation, and the higher the share of these fatty acids in membrane lipid pool, the greater the need for their antioxidant protection [6,7]. In addition, as described in Chapter 3, prenyllipid antioxidants themselves have an impact on the physicochemical properties of the membrane, so they may be involved in fluidity adjustment. In the case of carotenoids, their role in photoprotection is also important. Finally, prenyllipids may play a role in cold stress signalling. Literature data concerning the role of these compounds in the response to cold stress are reviewed in the next chapter.

5. Prenyllipid antioxidants in cold stress response

The positive role of Toc in the response to low temperature was reported for the model cyanobacterium Synechocystis sp. strain PCC 6803 [206]. This microorganism is insensitive to low temperature (5 °C) in darkness, but loses viability when cold-treatment is accompanied by the exposure to light (100 μmol photons m^-2s^-1). Preconditioning at 15 °C greatly improved the tolerance of Synechocystis sp. PCC6803 to cold and light. The α-Toc content in acclimated cyanobacteria was about 25% higher compared to non-acclimated ones. Subsequent exposure to 5 °C under light resulted in the decrease in α-Toc level, however it was less pronounced in preconditioned Synechocystis, compared to non-preconditioned cyanobacteria. After 8 days of stress treatment the higher level of α-Toc was accompanied by the lower level of lipid hydroperoxides in cold-acclimated blue-green algae compared to non-acclimated ones. Toc-deficient mutants slr1736, slr1737, slr0089, and slr0090 almost completely lost the ability to acclimate to low temperature plus light, while no differences or only slight differences were observed compared to wild type when mutants were incubated at 5 °C in darkness. In mutants slr1737 and slr0089, examined in this respect, exposure to low temperature under light resulted in a pronounced increase in the content of lipid hydroperoxides. Complementation of the slr0089 mutation restored the ability to stress acclimatisation. The results obtained suggest that the role of α-Toc is to provide antioxidant protection under combined low temperature and light [206]. Analysis of prenyllipid content in eukaryotic algae showed that snow and permafrost green algae accumulated high amounts of α-Toc [207,208].

Tocs are important for antioxidant protection under low temperature and high light, also in higher plants [209]. When leaf discs of A. thaliana, wild type and mutant vte1, were exposed to 1000 μmol photons m^-2s^-1 and 10 °C for 30 h, progress in lipid peroxidation, accompanied by a decrease in Chl content, was observed in mutant, while in wild type Chl degradation was less pronounced and increased lipid peroxidation did not occur. However, pronounced pigment bleaching and fatty acid degradation were not observed in whole mutant plants exposed to long-term stress (7 days, 1000 μmol photons m^-2s^-1 and 8 °C), suggesting compensatory mechanisms for Toc deficiency. It was shown that at low temperature under high light, the mutant vte1 accumulated more Zx than the wild type, which was accompanied by the enhanced efficiency of NPQ induction. The double mutant vte1npq1, lacking both Toc and VDE, showed a significant decrease in the maximum quantum yield of PS II, pigment bleaching, and enhanced lipid peroxidation, compared to the control and single mutants vte1 and npq1, when grown under high light at low temperature. These results showed that α-Toc and Zx may complement each other under low temperature and high light stress [209]. Maize seedlings grown at 15/13°C day/night since 6^th day after germination up to the three leaves stage contained 2.2-fold more α-Toc compared to seedlings grown at 25/22°C day/night. Cold-acclimated seedlings also had 25% more lutein and more than 2 times larger pool of violaxantin cycle pigments, which was highly de-epoxidised (Zx and Ax constituted half of the pool, whereas Vx was dominant in seedlings grown at normal conditions). They also displayed more efficient NPQ induction than non-stressed plants. Subsequent exposure to 1000 μmol photons m^-2s^-1 at 5 °C resulted in a less pronounced decrease in Chl, lutein, β-carotene, ascorbate, and glutathione in acclimated seedlings compared to non-acclimated ones [210]. The contents of α-Toc and total carotenoids increased approximately 2 times, and the de-epoxidised xanthophylls accumulated in the leaves of cold-hardened (5 weeks of growth at 4°C) winter rye (Secale cereale) when compared to non-hardened plants grown at 22 °C. Subsequent exposure to 500 μmol photons m^-2s^-1 at 4°C caused faster decrease in maximum quantum yield of PS II in non-acclimated plants [211]. The study on chilling-tolerant and chilling-sensitive genotypes of maize (Zea mays) showed that tolerance to low temperature might be correlated with an increase in α-tocopherol level, as well as with an increase in glutathione content and glutathione reductase activity [212]. The experiment on wheat cultivars showed that during cold acclimatisation (3 weeks at 5 °C, 130 μmol photons m^-2s^-1, photoperiod 8/16 h day/night), frost-tolerant cultivars accumulated more Tocs (all vitamers were present and α-Toc was dominant form) and β-carotene in leaves [213]. Transgenic tobacco (Nicotiana tabacum), accumulating high amounts of tocotrienols in leaves, exposed for 4 d to high light stress (700 μmol photons m^-2s^-1, 14/10 h photoperiod) at 10°C was able to maintain higher maximum quantum yield of PS II and higher content of Chls and carotenoids when compared to the wild type. The transgenic plants also showed less pronounced lipid peroxidation [214]. The Toc-deficient rice mutant sgd1-2 (the SGD1 gene is a homologue of VTE2) was hypersensitive to cold stress (3 days at 4 °C). The cold-treated mutant showed a more pronounced increase in proline level, compared to wild type plants, but this antioxidant was unable to compensate for Toc deficiency and provide survival after stress recovery [215]. On the contrary, exposure of Eucalyptus gunnii to 12 or 4°C caused a decrease both in the expression of EgSXD1 gene and α-Toc content [216].

An increase in the content of prenyllipid antioxidants under stress conditions is a common phenomenon, and cold stress is no exception in this regard [4]. Bergmüller et al. [217] observed a 2-fold increase in the content of Tocs in A. thaliana exposed to 4°C for 5 days. Interestingly, while α-Toc was a dominant vitamer in non-stress conditions, an increase in the share of γ-Toc in the leaf chromanol pool occurred under cold, heat (35°C), or high light (850 μmol photons m^-2s^-1) treatments. In both high light- and low temperature-exposed plants γ-Toc constituted about 25% of total Tocs. Slightly more of the other two vitamers were present in cold-treated Arabidopsis compared to high light-treated plants [217]. Experiments carried out for the sake of the Ph.D. thesis of the author of the present review showed that 14 days of exposure to high light or cold resulted in a significant increase in Toc content in the leaves of A. thaliana (Table 3). The sum of Tocs in high light- and low temperature-exposed plants was similar, however the share of γ-Toc in the chromanol pool was significantly higher in cold-treated plants, i.e., 28% versus 8% in high light-treated and 3% in control plants. Stress exposure also led to an increase in the content of α-TQ in leaves, as a result of enhanced oxidation of α-Toc. Unfortunately γ-TQ content was not assessed in this experiment [218]. However, both α-TQ and γ-TQ were shown to accumulate in runner bean (Phaseolus coccineus) seedlings after 12 h of exposure to low temperature (keeping the leaves on ice in darkness), followed by 3 h of high light exposure (750 μmol photons m^-2s^-1). Exposure to high light alone did not cause such an effect. It is worth to mention here that the young leaves of the runner bean contained unusually high level of γ-Toc [219]. An increased share of γ-Toc in total Tocs was observed in some species of plants that grow in the harsh climate of Patagonia [220]. It is an interesting question, if accumulation of γ-Toc is an effect of its slower or less efficient methylation, or the specific functions of this vitamer in cold-stressed plants.

Exposure of alfalfa (Medicago sativa) to 10 °C for 24 h in darkness led to an increase in α-Toc content, while during recovery at 25 °C the level of this antioxidant decreased [221]. Lepidium sativum seedlings grown for 5 days at 10 °C contained much more γ-Toc and PC-8, and slightly more α-Toc than plants grown at 22 °C [222]. The content of α-Toc increased during cold stress in varieties of wheat, Triticum aestivum and Triticum durum. The 6-day-old seedlings were transferred from normal conditions (24/16°C day/night) to low temperature conditions (5/-5°C day/night) and kept there for 6 days. Taking into account other prenyllipids, low temperature treatment caused an increase in the xanthophyll content and slight decrease in β-carotene content [223].

A high α-Toc content was found in several species of mosses that grow in Antarctica [224]. The 1.4-fold increase in α-Toc content during autumn and the persistence of an elevated level of this antioxidant during winter was observed in the evergreen shrub Ephedra monosperma that grows in Central Yakutia. These changes were accompanied by changes in carotenoid content, i.e., a decrease in β-carotene, increase in lutein, accumulation of rhodoxanthin, and de-epoxidation of Vx to Ax and Zx. Photoprotective xanthophylls were kept at elevated levels during winter [225,226]. The leaves and rhizomes of the seagrass, Cymodocea nodosa, contained more α-Toc in winter and spring than in summer [227]. In Mediterranean evergreen species an increase in α-tocopherol content accompanied by night retention of de-epoxidised xanthophylls was observed in response to an abrupt period of exceptional cold weather. The accumulation of Zx and Ax was correlated with a sustained decrease in maximum quantum yield of PS II [228]. The high mountain plant Soldanella alpina, collected at its natural sites, contained high levels of α-Toc and violaxanthin cycle pigments. The amounts of these compounds decreased when the plants were transferred to controlled conditions and grown at 22°C for 3 weeks. Interestingly, when S. alpina was grown for 3 weeks at 6 °C, α-Toc level decreased, but carotenoids remained at levels similar to those measured in plants growing in the mountains [229]. High levels of α-Toc were also detected in alpine grazing plants from Norway [230]. Desiccation-tolerant overwintering fern species contained more α-Toc, γ-Toc and violaxanthin cycle pigments than non-tolerant species. They also displayed an increased de-epoxidation ratio. All tested species accumulated Zx and Ax as a response to controlled freezing. These xanthophylls were shown to accumulate during natural freezing in the field at night, which was accompanied by a decrease in maximum quantum yield of PS II [231].

An interesting aspect of Toc functions under low temperature conditions is related to its role in signalling. As mentioned in Subchapter 3.1, Tocs are involved in the regulation of assimilate transport [85]. Disturbances in differentiation of plasmodesmata, their occlusion by callose, death of vascular parenchyma cells, and impaired sucrose export from leaves were observed in the sxd1 mutant of maize lacking tocopherol cyclase [84,85,86]. A photoassimilate export-defective phenotype and vascular-specific callose deposition were also observed in transgenic potato, in which the expression of StSXD1 was silenced using the RNAi method [232]. The Toc-deficient vte1 and vte2 mutants of A. thaliana did not display such a phenotype under optimal growth conditions; however, it appeared under low temperature [233]. Cold-treated mutants showed stunted growth, lower yield, callose deposition, and impaired carbohydrate export from leaves. Exposure to 7.5°C for 4 weeks resulted in about 5-fold increase in total Tocs in control plants. During this time, the content of Chls decreased transiently, while the carotenoid content slightly increased. A progressive decline in Chls and carotenoids was observed in mutants, which was accompanied by accumulation of anthocyanins. The fact that the observed changes were independent of light level and occurred in the absence of photoinhibition or lipid peroxidation let the authors to conclude that the mechanisms involved were independent of antioxidant functions of Toc [233]. Further research on the vte2 mutant showed that its lipid profile was different compared to the wild type, i.e., the cold-treated mutant contained a lower content of linolenic acid and a higher level of linoleic acid. The change in lipid composition turned out to be the result of the reduced conversion of dienoic to trienoic fatty acids in the endoplasmic reticulum (ER). Therefore, it was postulated that Toc plays a role in modulation of PUFA metabolism [234]. The cold-induced phenotype was suppressed in the double mutant vte2fad2 and to a lesser extent in the mutant vte2fad6, defective in both Tocs and fatty acid desaturases, playing a role in the conversion of monoenoic to dienoic fatty acids [234]. The suppression of the cold-induced phenotype of the vte2 mutant was also observed in plants that carry mutations in transporters responsible for the transport of lipids from the ER to the plastids [235].

The role of isoprenoid quinones in cold acclimatisation and resistance was examined to a lesser extent. Similarly to α-Toc, PQ and PQH₂ levels increased in autumn in E. monosperma. The beginning of exposure to a freezing average daily temperature resulted in a depletion of the PQ pool by 18%. Even through, the content of PQ_tot in the winter months was approximately 1.5-fold higher than that measured at the end of July [226]. The content of PQ in chloroplasts isolated from rye (Secale cereale) grown at 5 °C was approximately 1.6-fold higher than that of the control grown at 20 °C [236]. Pea plants grown at 6 °C showed a higher PQ content normalised to Chl compared to plants grown at 18°C. However, such an effect was not observed in cucumber (Cucumis sativus), oats (Avena sativa) and Lolium tremulentum. In L. tremulentum, the PQ content was decreased in plants grown at 6 °C when compared to those grown at 18°C [237]. In Scots pine (Pinus sylvestris), the PQ content decreased in autumn and increased in late spring, which was correlated with changes in Chl content. These results suggest that the changes of the PQ pool in this species play a role in the adjustment of photosynthesis rather than in antioxidant protection [238]. The experiments concerning the UQ response to low temperature are scarce. A comparison of the cold response (3 days at 10°C followed by 3 days at 5°C) of chilling-sensitive mung bean (Vigna radiata) and chilling-tolerant pea (Pisum sativum) seedlings showed that the UQH₂, UQ, and total ubiquinone (UQ_tot) content decreased in the mung bean, but not in the pea in response to the temperature treatment [239]. Therefore, the UQ content seems to be associated with the ability of metabolic adjustment.

In addition to experiments that measured both Tocs and carotenoids, there were many others that evaluated only the carotenoid content. Cold acclimatisation is usually accompanied by an increase in the carotenoid level. Exposure of the model green microalga Chlamydomonas reinhardtii, naturally living in moderate climate, and the snow alga Chlamydomonas nivalis to 4 °C under 100 µmols photons m^-2s^-1 for 72 h resulted in an increase in total carotenoid content compared to controls grown at 22 °C. This effect was more pronounced in C. nivalis [240]. C. reinhardtii grown at 10, 15, and 20 °C contained more carotenoids per cell than the algae cultivated at 25 °C [241]. In the red alga Betaphycus gelatinus 2 h of incubation at 15 °C was enough to cause a 2-fold increase in the total carotenoid content when compared to the control kept at 27 °C [242]. Stramenopile microalgae Nannochloropsis oceanica grown for 3 days at 15 °C showed a 2-fold increase in the content of total carotenoids, β-carotene and Vx, and approximately 1.5-fold increase in Zx level, compared to algae grown at 30 °C [243]. The cold acclimatisation of spinach plants (Spinacia oleracea L.) resulted in a 20% increase in total carotenoids normalised to Chl compared to the control. In this experiment, 5-week-old plants were kept at 18 °C or cold-acclimatised by gradual decrease of temperature from 18 to 1 °C for 10 days under the same light regime, 260-300 µmols photons m^-2s^-1 8/16 day/night [244]. A gradual increase in total carotenoids was observed in 2-week-old Calendula officinalis seedlings transferred to 4 °C for 5 days, while in control seedlings grown at 25 °C the level of these pigments remained unchanged [245]. Total carotenoid content in 8-week-old kale (Brassica oleracea var. acephala) exposed to 8 °C for 24 h was approximately 15% higher than that of the control kept at normal growth temperature, 21 °C [246]. Exposure of 4-week-old bell pepper (Capsicum annuum) plants to low temperature (15/10 °C day/night), UV-B (72 kJm^-2, 6 h per day) or low temperature plus UV-B for 7 days led to an increase in carotenoid content. The increase was more pronounced when both stress factors were applied simultaneously [247]. The silencing of the CaLCYB gene encoding lycopene β-cyclase of C. annuum resulted in a decrease in α- and β-carotene, Vx, Zx, and lutein content, and a decrease in tolerance to low temperature (15/5 °C day/night versus control conditions 25/22 °C), assessed visually, and by measuring markers of oxidative stress and maximum quantum yield of PS II [248].

An increase in the amount of violaxanthin cycle pigments and the share of de-epoxidised xanthopylls in total carotenoid pool is a common element of cold acclimatisation. This response enables a more efficient NPQ and thus decreases the risk of ROS formation. Potato leaves pre-illuminated (1 h, 900 µmols photons m^-2s^-1 at 23 °C) to provide Vx de-epoxidation, and then exposed to 2000 µmols photons m^-2s^-1 at 3 °C, showed less pronounced lipid peroxidation than non-preilluminated leaves exposed to high light at low temperature [249]. Low temperature (5°C) and high light (1500 µmols photons m^-2s^-1) acted synergistically to induce an increase in violaxanthin cycle pigments in barley (Hordeum vulgare) seedlings exposed to stress conditions for 5 days [250]. Koroleva et al. [251] compared the activity of the xanthophyll cycle and NPQ in the chilling-sensitive domestic tomato, Lycopersicon esculentum cv. Abunda and two relatively chilling-tolerant lines of wild tomato (Lycopersicon peruvianum). Plants grown at 20-27 °C were transferred to low temperature and high light conditions (10 °C, 300 µmols photons m^-2s^-1, 12/12 day/night), and measurements were carried out after 2 weeks of stress exposure. For all genotypes tested, stress caused a substantial increase in the content of lutein, Nx, and violaxanthin cycle pigments normalised to Chl in leaves, which was associated by increased efficiency of NPQ induction, compared to the non-stressed control. In L. esculentum and L. peruvianum line LA 385 cold acclimatisation resulted in the increased activity of the xanthophyll cycle [251]. In another study on tomato cold tolerance, the cold response was compared in L. esculentum cv. Abunda, low-altitude wild species L. pimpinellifolium, and three high-altitude wild species L. peruvianum, L. hirsutum, and L. chilense. Five-leave-stage plants grown at 25/20 °C day/night, 225 µmols photons m^-2s^-1, 14/10 day/night were transferred to 10 °C, 75 µmols photons m^-2s^-1, 14/10 day/night for 2 weeks. In domestic and low-altitude species, cold exposure caused an increase in the content of violaxanthin cycle pigments normalised to Chl, whereas the de-epoxidation state increased in high-altitude species. Both responses resulted in higher Zx levels in the leaves of all cold-treated plants. The lutein level increased only in stress-exposed L. pimpinellifolium and L. hirsutum [252]. Ramalho et al. [253] examined the cold response in three coffea species, C. arabica, C. canephora and C. dewevrei, during a 20-day period of gradual temperature decrease, from 25/20 °C to 15/10 °C (day/night), during a subsequent chilling treatment (3 days with 15/4 °C, when 4 °C was kept at night and for the first 4 h of the light phase), and after 6 days of rewarming (25/20 °C day/night). C. arabica turned out to be more cold tolerant than two other species, which was correlated with a higher content of total carotenoids, lutein, Nx, and Zx observed during the whole experiment. This species showed the most pronounced increase in lutein and β-carotene content, during cold acclimatisation and during 15/4 °C treatment, respectively [253]. The comparison of two pepper cultivars, chilling-sensitive and relatively tolerant to chilling, showed that when five leaf stage plants, grown at 25/20 °C day/night, were transferred to 14 °C or 25 °C, both under 250 µmols photons m^-2s^-1, 12/12 day/night, for 5 days, the content of β-carotene and lutein was about 15-20% higher, while the content of total carotenoids was 20-30% higher in cold-acclimated plants than in non-acclimated ones. The cold-acclimated chilling-tolerant cultivar showed a 65% increase in violaxanthin cycle pigments compared to a 50% increase in the cold-acclimated chilling-sensitive cultivar; it also contained more Ax and Zx. Subsequent exposure of plants to 1200 µmols photons m^-2s^-1 at 4 °C caused more efficient induction of NPQ and more pronounced de-epoxidation of violaxanthin cycle pigments in cold-acclimated plants compared to non-acclimated plants, while the final values of the NPQ parameter and the ratio (Ax + Zx)/(Vx + Ax + Zx) were higher in the cultivar tolerant to chilling compared to chilling-sensitive one [254]. Cold acclimatisation (2 months at 9°C) of Oxyria digyna, naturally growing in arctic and alpine habitats, resulted in an increased capacity for Vx de-epoxidation during 20 min of incubation under 2000 µmols photons m^-2s^-1 at 4 °C, compared to non-acclimated plants grown at 18-25°C [255].

The role of carotenoids in cold response and cold tolerance was also studied in monocotyledon crop species. 3-day exposure of 3-week-old maize plants (Zea mays) to 15 °C resulted in a 1.6-fold increase in the content of violaxanthin cycle pigments and the de-epoxidation of their pool, compared to plants grown at 25/20 °C day/night [256]. P. Haldimann checked the cold response of three chilling-tolerant and three chilling-sensitive genotypes of maize. Five-day-old seedlings grown at 24/22 °C day/night were kept at this temperature or transferred to 14/12 °C, and then grown until the three leaf stage. Two light regimes were tested, 200 and 600 µmols photons m^-2s^-1. Cold-exposed plants showed an increase in carotenoid content normalised to Chl and the effect was more pronounced in the chilling-sensitive genotypes than in the tolerant ones, particularly under high light. Taking into account particular carotenoids, all genotypes showed an increased content of lutein, Nx, and violaxanthin cycle pigments, but a decreased content of β-carotene normalised to Chl, especially under high light. The increase in both violaxanthin cycle pigments and the share of Zx in their pool was more pronounced in stressed chilling-sensitive lines compared to chilling-tolerant genotypes [257]. When 2-week-old seedlings of two cold-tolerant japonica rice genotypes grown at 28 °C, 300 µmols photons m^-2s^-1, 16/8 day/night were exposed to 10 °C for 48 h, they showed an increase in the content of lutein and increased de-epoxidation state of violaxanthin cycle pigments [258]. In etiolated barley seedlings, grown at 20 or 5 °C and exposed to light (250 and 800 µmols photons m^-2s^-1 for control and 50 and 250 µmols photons m^-2s^-1 for cold-stressed plants), the most pronounced increase in the content of violaxanthin cycle pigments was observed for plants exposed to 250 µmols photons m^-2s^-1 at 5 °C. In addition, only in this series there was an increase in lutein and Nx. The seedlings etiolated at 5 °C under 250 µmols photons m^-2s^-1 displayed a higher value of the qN parameter representing the NPQ efficiency compared to the seedlings etiolated at 20 °C under the same light intensity [259].

An increase in the content of violaxanthin cycle pigments is often observed during frost-hardening in species adapted to a cold climate. Experiment in cold acclimatisation of the coniferous tree Picea glehnii, a cold-tolerant species of the boreal zone, showed that the exposure of 4-year-old plants to 15/10 °C day/night for 10 days and then to 5/0 °C day/night for another 10 days resulted in an increase in the content of violaxanthin cycle pigments, in particular Zx, and a slight, but not statistically significant increase in lutein and β-carotene, when compared to control plants grown at 25/20 °C day/night. Subsequent exposure of P. glehnii to 0 °C for 2 weeks caused an increase in violaxanthin cycle pigments, mainly Zx, in non-acclimated plants, while the content of these xanthophylls did not change in acclimated plants [260]. Three conifer species, Pinus ponderosa, Pseudotsuga menziesii and Picea pungens, contained higher levels of lutein and violaxanthin cycle pigments, and lower levels of α-carotene during winter. In addition, the de-epoxidation of Vx at midday was more pronounced in winter than in summer. Ax and Zx were retained at high levels overnight during winter. The predawn conversion state of the violaxanthin cycle was related to day to day variation in temperature, being more de-epoxidised on colder days [261]. A similar effect was observed in Scots pine, where the amount of lutein and violaxanthin cycle pigments increased 2-fold in winter, α-carotene level decreased, and the share of de-epoxidised xanthophylls remained high. The timing of seasonal changes indicated that de-epoxidation of xanthophylls correlates with the winter quenching of Chl fluorescence [262]. An independent study on this species by the Sofronova group showed a pronounced increase in total xanthophylls, lutein, and Zx, accompanied by pronounced decrease in β-carotene and slight decrease in Nx in autumn [263]. An increase in the content of lutein and violaxanthin cycle pigments, and the retention of de-epoxidized xanthophylls during winter was also reported for Taunton yew (Taxus x media). In this plant, an increase in the content of these pigments was more pronounced in sun leaves compared to shade leaves [264]. The amount of violaxanthin cycle pigments increased in autumn in both sun and shade leaves of Cryptomeria japonica. However, rhodoxanthin accumulation and retention of de-epoxidised xanthophylls at night were observed during winter only in sun leaves [265]. The evaluation of pigment composition in Qilian juniper (Sabina przewalskii) growing at its typical sites, wetter and drier, showed that an increased de-epoxidation state of xanthophylls occurred in winter, but not in summer, in plants growing at wet site, while high de-epoxidation of the violaxanthin cycle pool was sustained during whole year in plants from the dry site. On the other hand, increased level of rhodoxanthin in winter was reported for juniper growing at both sites [266]. An increase in the content of violaxanthin cycle pigments normalised to total Chl and increased de-epoxidation state of their pool in winter, as well as the retention of a highly de-epoxidised state of the pool during winter nights, was also observed in broad-leafed evergreen plants. In perennial shrub, Mahonia repens, this effect was the most pronounced in sun-exposed leaves and least pronounced in shade leaves [267]. An increase in the amount of violaxanthin cycle pigments, lutein and Nx in the autumn, accompanied by a decrease in β-carotene, as well as the retention of de-epoxidised xanthophylls during nights in winter were reported for two elevational ecotypes of Rhododendron catawbiense [268]. Analysis of pigment content and antioxidant content in winter-red (anthocyanic) and winter-green (acyanic) angiosperm evergreen species showed that anthocyanins likely function as a photoprotective strategy alternative to the one based on increased content of violaxanthin cycle pigments during winter [269]. The results of the experiment on two dominant arctic shrub species, Salix pulchra and Betula nana, support the hypothesis that xanthophyll cycle activity plays an important protective role in plants growing in harsh conditions. The high retention of de-epoxidised xanthophylls and saturation of the conversion of the violaxanthin cycle at low-light intensities were related to the high efficiency of NPQ measured in both species [270]. Analysis of the cold response of two ecotypes (Antarctic and Andes) of Colobanthus quitensis, which were cold acclimated at 4 °C for 3 weeks and then exposed to 1600 or 100 µmols photons m^-2s^-1 for 2 h, showed that in both ecotypes, in non-acclimated plants grown at 13-15 °C and cold-acclimated plants, high light exposure caused an increase in lutein content. The cold-acclimated Antarctic ecotype exposed to high light exhibited a lower de-epoxidation state of the violaxanthin cycle pigments compared to the cold-acclimated Andes ecotype, whereas such a difference was not observed for non-acclimated plants [271]. Analysis of violaxanthin cycle pigments and xanthophyll cycle activity in Antarctic plants, Polytrichum juniperinum (moss), and Deschampsia antarctica (grass) revealed that the total pool of xanthophyll cycle pigments was in the upper range of the values reported for most plant species, and that there was a strong conversion of Vx to Zx during the day. Overnight retention of Zx was not observed [272]. Antarctic moss species, Bryum pseudotriquetrum, Ceratodon purpureus, and Schistidium antarctici, showed sustained high levels of xanthophyll pigments, especially the plants growing at exposed sites [273]. An increase in carotenoids to Chl ratio in Ceratodon purpureus growing at light-exposed sites when compared to plants growing at shaded sites was also observed by other scientists. In particular, the Vx content was higher in moss growing in sun-exposed sites [274]. Another antarctic moss, Bryum argentum, contained relatively high amounts of violaxanthin cycle pigments. Sun-exposed moss contained more carotenoids than shade forms [275].

Recent studies on carotenoids and cold stress involve their biosynthesis and related signalling. Exposure of maize seedlings to 10 °C caused an increase in the expression of genes that encode phytoene synthase (ZnPSY2 and ZmPSY3), phytoene desaturase (ZmPDS), and violaxanthin deepoxidase (ZmVDE). On the other hand, the expression of ZmCRTISO, which encodes carotene isomerase, was lower in cold-treated seedlings compared to the control grown at 25 °C [276]. Li et al. [277] identified five genes that encode phytoene synthases in tobacco and examined their expression patterns. Among them, NtPSY1 was significantly induced by low temperature treatment. Overexpression of this gene enhanced β-carotene accumulation and improved cold tolerance in transgenic plants, assessed as survival rate after 2 weeks of growth at 10 °C [277]. The transgenic tobacco overexpressing tomato gene LeLUT1, encoding lycopene ε-synthase, had increased lutein level. When transgenic plants were exposed to low temperature (4 °C), they showed enhanced NPQ, lower levels of oxidative stress markers and membrane leakage, higher values of maximum quantum yield of PS II, net photosynthetic rate, and total Chl content, as well as less suppressed growth than cold-treated wild type [278]. Analysis of the expression of genes belonging to the CCD family in Forsythia suspensa showed that four of the five genes belonging to the 9-cis epoxycarotenoid dioxygenases (NCED) subfamily were significantly up-regulated under cold stress. Their products were postulated to participate in the synthesis of ABA under cold stress conditions [279].

6. Conclusions and further perspectives

The impact of low temperature on membrane properties, as well as the occurrence of oxidative stress in cold-exposed plants, especially under high light, are phenomena well documented in the scientific literature. The role of prenyllipid antioxidants, in particular carotenoids and Tocs, in cold acclimatisation has also been widely described. However, the vast majority of the literature focusses on the accumulation of these compounds as a response to chilling stress, the role of carotenoids in photoprotection, and the antioxidant action of prenyllipids. An interesting question would be to determine the exact impact of carotenoids and isoprenoid chromanols and quinones on the physicochemical properties of membranes whose composition is similar to that present in nature, in a wide range of temperatures. More experiments are needed on the roles of PQ, UQ, or phylloquinone in the response to cold stress, because these prenyllipids are examined to a lesser extent compared to Tocs and carotenoids. The very interesting research direction is the deciphering of the regulation of prenyllipid biosynthetic pathways in response to low temperature. The next step would be to obtain more lines of transgenic plants that overexpress genes crucial for the enhancement of the synthesis of carotenoids and Tocs to check if accumulation of these compounds would improve cold tolerance.