Because of their sessile nature, plants are unable to avoid fluctuating environment conditions like high light, ultraviolet radiation, drought, salt stress, heat, cold or flooding. Upon certain threshold of these changes, plant cells can no longer maintain proper metabolic processes and programmed cell death (PCD) is induced.
PCD is an essential cell suicide process in animals, yeast and plants. In multicellular organisms, it plays an important role in the cell homeostasis maintenance, tissue specialization, removing of damaged or infected cells and acclimatory response. In contrast to necrotic death, which proceeds via swelling, lysis and leakage of cell content, PCD is a highly regulated and organized process. This controlled disassembly of cell involves the condensation, shrinkage and fragmentation of both cytoplasm and nucleus and DNA laddering. Furthermore, while PCD can occur during development and is regulated by complex mechanisms, necrosis does not require the activity of proteases nor nucleases and is not associated with signal transduction pathways .
There are two main categories of PCD in plants: developmentally- and environmentally-induced PCD. The first one is a genetically encoded process which plays a crucial role in the development of some tissues and organs. It is involved in tracheary elements formulation during xylem differentiation. Tracheary elements are long cells that transport water and mineral salts, and serve as a structural support in vascular plants. Their formation occurs after secondary wall synthesis and begins with a collapse of the central vacuole and release of lytic enzymes, followed by degradation of cellular content . Another example of developmentally-induced PCD is a formation of unisexual flowers in monoecious species (e.g. maize), bearing generative organs of both sexes on the same plant. Sex determination in these species involves the developmental arrest of one of the organ primordia - either the female or male within a bisexual ﬂoral meristem . The production of complex leaf shapes also frequently employs PCD. Such remodeling of leaf blades occurs in
Developmental PCD is induced by internal factors and occurs at defined time and in particular plant tissue. On the contrary, environmentally-induced PCD is triggered by different stimuli ranging from pathogen infection to environmental factors . During infection of plant leaves by pathogens, a specific gene-for-gene (avr–R) interaction triggers defense responses. Upon such plant-microbe interaction, cell death takes a form of so-called hypersensitive response (HR) and includes a burst of reactive oxygen species (ROS). HR leads to the formation of a lesion which is clearly delimited from surrounding healthy cells and thus prevents the spread of pathogen throughout the plant tissue. Certain mutations in many plant species have been demonstrated to cause spontaneous, HR-resembling lesions, which suggests that this type of cell death is under a genetic control. Such lesion mimic mutants are divided into two groups: related to the initiation of PCD (inappropriate induction of PCD and formation of localized lesion spots) or propagation (inability to stop PCD once it has been initiated). Both these groups of mutants are currently widely investigated since they can provide insight into the general mechanism of PCD in plants [8–12]. The existence of these two classes suggests that genetically distinct processes underlie the lesion formation: the initiation of cell death and its spread to surrounding cells as well as the existence of communication signals between dying and healthy cells in determining the lesion size.
In natural habitats, plants are constantly exposed to a variety of environmental stresses that can lead to the disturbance in cellular homeostasis and consequently limit crop yield. Programmed cell death is a fundamental cellular process associated with the defense responses to abiotic stimuli such as excessive irradiation, ozone, ultraviolet radiation, heat, cold, drought or flooding. One of the example factor triggering PCD is hypoxia, a condition in which plant is deprived of oxygen supply. In response to waterlogging and lower O2 concentration in the ground, cortex of the root can form aerating tissue called aerenchyma . The internal air spaces are generated through PCD and facilitate gas diffusion from aerial organs to waterlogged roots . Although it is unfavorable for biomass production, the selective death of cells and tissues under abiotic stresses eventually provides survival advantages for the whole organism. At the organismal level, PCD helps to maintain tissue and organ homeostasis, enables developmental adaptation and nutrient resorption from dying cells thus increases the probability of survival. It also leads to the signals transduction from cells undergoing PCD to healthy, not-affected cells and triggers stress tolerance and acclimation to adverse conditions [6,15].
2. Hallmarks and the regulation of programmed cell death
While the cascade of events and molecules regulating PCD have been already well described in animal cells, mechanisms underlying plant PCD remain still inexplicable. Therefore, numerous studies in plants rely on the comparison of PCD mechanisms to animals. Apoptosis (well-studied form of animal PCD) features in cell shrinkage, chromatin condensation, cleavage of DNA (called DNA laddering) and nuclear fragmentation. The mechanism of PCD depends on a family of cysteine proteases called caspases that cleave their target proteins after aspartic acid residues. Caspases are synthesized in the cell as inactive precursors (procaspases). Once activated, caspases cleave and activate other procaspases which results in a self-amplifying cascade. They can also cleave other proteins such as nuclear lamins or proteins that hold DNA-degrading enzymes in inactive form, releasing DNases to cut DNA. The destructive protease cascade is irreversible, therefore caspase activity needs to be tightly controlled. Procaspase activation is induced by the release of electron carrier protein - cytochrome c from mitochondria to the cytosol. The family of Bcl-2 proteins regulates the activation of programmed cell death. Some members of this family (e.g. Bcl-2) block the release of cytochrome c, inhibiting apoptosis. Others (e.g. Bax and Bak) act as PCD inducers, promoting cytochrome c leakage. IAP (inhibitor of apoptosis) proteins are another family involved in apoptosis regulation as they bind to some procaspases, preventing their activation or to caspases, inhibiting their activity. Proteins that block IAPs are released together with cytochrome c which increases the efficiency of cell death process .
Many hallmarks of plant PCD seem to be similar to animals such as cytoplasm shrinkage, chromatin condensation and DNA cleavage, mitochondrial swelling, disruption of organelles and plasma membrane collapse . The major difference in executing PCD between animals and plants lies in the process of removing the cell content after its death. While in animal cells, removal action is undertaken by other cells to avoid the activation of inflammatory response, in plants there is a leakage of the cell content into the apoplast and remains are not engulfed by surrounding cells . Moreover, plants exhibit some distinctive features of PCD that result from the presence of chloroplasts and the significance of vacuoles [18,19]. Plant vacuoles represent important storage organelles that are the repository of hydrolytic enzymes such as proteases, lipases and nucleases. Vacuoles are therefore postulated to play a role in the turnover of organelles and cytoplasm during autophagy as a part of clean-up system for dying cells. The component of this system is a caspase-like protease - the vacuolar processing enzyme (VPE) which plays a crucial role in such PCD pathways as senescence, lateral root formation and hypersensitive response. Upon receiving pro-apoptotic signals, VPE activates hydrolases that execute the degradation of vacuolar membrane resulting in the release of hydrolytic enzymes and subsequent degradation of cell content .
Chloroplasts are strongly suggested to be key players during cell death responses as they constitute an important source of defense signaling molecules such as ROS, reactive nitrogen species (RNS) and defense hormones like salicylic acid (SA) and jasmonic acid (JA). The oxidative burst is one of the earliest and most common plant response to abiotic and biotic stimuli . The application of chloroplast-targeted, ROS-generating herbicides such as methyl viologen (paraquat) induces cell death with the typical apoptotic traits .
Some of key proteins controlling animal cell death such as the Bcl-2 family and caspases have been proven to be not conserved in plants. It suggests that plants have developed some unique mechanisms of PCD . Although orthologs of caspases have not been found in plants based on the sequence similarity, several studies using caspase-speciﬁc peptide inhibitors suggested the presence of caspase-like proteases (metacaspases) . These caspase inhibitors have been demonstrated to prevent chemically-, UV- or HR-induced PCD [23–25] indicating that caspase-like proteins are indeed involved in the regulation of PCD in plants. Metacaspases (MC) differ from animal caspases in their substrate specificity as they cleave proteins after arginine or a lysine residues. Nine predicted metacaspase-encoding genes have been found in
Although no orthologues of Bcl-2 family genes (
Numerous signals are constantly integrated by the cell to decide whether to enter or not the cell death pathway. Different plant hormones are involved in the regulation of cell death under unfavorable conditions. One of the most important is SA which is intensively produced in cells after pathogen infection or various abiotic stresses . Many lesion mimic mutants have constitutively elevated levels of SA . At high concentrations, SA functions as a cell death inducer in cooperation with other signals. It can be also transported beyond the site of synthesis, acting as a signaling molecule and mediating systemic acquired resistance (SAR) - a whole-plant resistance response that prepares plant for another infection . The existence of SA-dependent generation of ROS and the feedback control of SA synthesis by ROS have been also demonstrated . SA and ROS have been proposed to work in a potentiation feedback loop which acts to amplify signals leading to cell death. Another cell death signaling molecule - nitric oxide (NO) has been also demonstrated to regulate key steps in SA biosynthesis during pathogen infection . Additionally, NO has been proven to cooperate with ROS and SA in inducing cell death . Other phytohormones regulating cell death under stress conditions are JA, gibberellic acid (GA), abscisic acid (ABA) and ethylene (ET). The latter is involved in the regulation of PCD during different developmental processes and responses to environmental stimuli. ET has been proven to participate in the formation of aerenchyma in roots under hypoxia . Antisense inactivation of the ET biosynthetic enzyme - ACC oxidase delays leaf senescence and cell death in tomato . Ethylene is also required for the continuation of ROS accumulation - external supply of ET during cell death increases ROS production and causes accelerated spreading of cell death . JA is a plant signaling molecule best known for its role in the wound response but it is also produced during wide range of biotic and abiotic stresses. It is involved in the inhibition of ROS- and ET-dependent lesion propagation . JA derivatives such as methyl jasmonate (MeJA) are also engaged in the regulation of plant immune responses . Upon exposure to stress, MeJA is produced and causes the activation of PCD through the induction of ROS generation, alterations in mitochondrial dynamics and photosynthetic collapse . Another phytohormone - GA has been proven to promote cell death in cooperation with ROS, whereas ABA delays GA-induced PCD. Such counteracting role of these hormones relates to their influence on the ROS-scavenging enzymes expression . ABA has been also shown to delay ET- and GA-induced cell death in rice epidermal cells . All these interactions between phytohormones and ROS indicate the complexity of PCD regulation. Overmyer and colleagues  suggested the following series of events during oxidative stress-triggered PCD. At the site of lesion initiation, the action of ROS is amplified. Increased ROS accumulation together with SA induces the cell death. During the initial process, JA signaling is hindered by SA and ET. Meanwhile, the burst of ET from the initial site disperses to surrounding cells, amplifies ROS production that promotes the lesion spread. This is the signal to induce competitive reactions to PCD. Cell death results in the production of JA which acts as a negative regulator of the oxidative cell death cycle. JA, through the suppression of SA biosynthesis/signaling and the attenuation of ET sensitivity, inhibits the lesion propagation.
During early events of hypersensitive response, ion fluxes are induced. Ca2+ influx caused by external hydrogen peroxide application has been demonstrated to be sufficient in triggering HR in soybean cells . Moreover, several cell death signaling proteins in plants exhibit a function associated with lipids. Two
3. Reactive oxygen species in plants
The signaling during PCD proceeds mainly through the regulation of reactive oxygen species [60,64,65]. ROS are produced continuously as by-products of various pathways localized in chloroplasts, mitochondria and peroxisomes. They can occur as free radicals: superoxide radical (O2•−), hydroxyl radical (•OH), perhydroxyl radical (HO2•), alkoxy radical (RO•) or in non-radical forms: singlet oxygen (1O2) and hydrogen peroxide (H2O2). Most abiotic stresses evoke the overproduction of ROS in plant tissues. Because of their high reactivity, ROS can cause damage of proteins, lipids, carbohydrates and nucleic acids, ultimately leading to cell death (Figure 2).
The single reduction of O2 results in the formation of O2•−. From O2•− other more reactive ROS like •OH or HO2• can be formed. The Haber-Weiss reaction generates hydroxyl radical from hydrogen peroxide and superoxide. In this reaction O2•− donates an electron to Fe3+, reducing ferric ion to ferrous:
Fe3+ + O2•−→ Fe2+ + O2
The second step of •OH formation is the Fenton reaction in which reduced form of iron (Fe2+) transfers electrons to H2O2:
Fe2+ + H2O2 → Fe3+ + OH− + •OH
O2•− can be also protonated to form HO2•. Furthermore, O2•− can react with another free radical species as NO• to generate peroxynitrite (OONO−) . Another form of ROS – singlet oxygen is the first excited electron state of O2 that originates when an electron is elevated to a higher energy orbital (Figure 3).
Very reactive 1O2 can be formed by the reaction of O2 with photo-excited chlorophyll. Inadequate dissipation of excess energy during photosynthesis can lead to the generation of chlorophyll triplet state. The Chl triplet state can react with 3O2 to yield 1O2 . The formation of 1O2 has extremely damaging effect on PSI and PSII and other components of photosynthetic machinery. The 1O2 lifetime has been measured to be approximately 3µs . During this time, some part of 1O2 fraction is able to diffuse over several hundred nm, reacting with proteins, pigments, nucleic acids and lipids . In
It has been estimated that 1 - 2% of O2 consumed by plants is sidetracked for ROS production in different subcellular compartments . Organelles such as chloroplasts, mitochondria or peroxisomes are major sources of ROS in plant cells since they exhibit an intense rate of electron flow and highly oxidizing metabolic activity. In chloroplasts, PSI and PSII are major sites where the singlet oxygen and superoxide radicals are generated. Under non-stress conditions, the electron from excited photosystem is transferred to NADP+, reducing it to NADPH. However, under various abiotic stresses, the electron transport chain (ETC) tends to be overloaded and a part of the electron flow is diverted from ferredoxin to O2, reducing it to O2•−. The photoreduction of O2 at PSI proceeds via Mehler reaction and produces O2•−, which is disproportionated to H2O2 and O2 with the use of superoxide dismutase. H2O2 is rapidly detoxified to H2O by the ascorbate peroxidase pathway (Figure 4A). Because of the electron flow from water in PSII to water in PSI that occurs in this process, it has been termed the water–water cycle . This cycle does not only scavenge O2•− and H2O2, but also generates a pH gradient across thylakoid membranes which enhances non-radiative dissipation of light energy by non-photochemical quenching (see later). Therefore, the water–water cycle is considered to function as a dissipatory mechanism of the excess energy [77,78].
H2O2 is also produced during a process that proceeds concurrently to the photosynthesis – photorespiration. During photosynthetic carbon assimilation, ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme (Rubisco) uses CO2 to carboxylate ribulose-1,5-bisphosphate (RuBP). CO2 uptake results in the formation of two molecules of 3-phosphoglycerate (3-PGA) that are utilized for biosynthetic reactions and the recycling of the RuBP acceptor molecule. However, Rubisco can also use O2 to oxygenate RuBP, forming one molecule of 3-PGA and one molecule of 2-phosphoglycolate (2-PG). The latter cannot be used for biosynthetic reactions and is considered as an inhibitor of the chloroplast function. Photorespiration functions to convert 2-PG back to 3-PGA and thus to recover carbon. It constitutes a series of reactions taking place in chloroplasts, peroxisomes, and mitochondria. 2-PG is dephosphorylated to glycolate in the chloroplast and transported to the peroxisome where it is oxidized to glyoxylate. O2 is the electron donor in this reaction, which results in H2O2 generation. Glyoxylate is transaminated to glycine which is transported to the mitochondrion, where two molecules of glycine are converted to serine and the remaining carbon and nitrogen are released as CO2 and NH3, respectively. The amine group is used to form a new glycine from glyoxylate and the resulting hydroxypyruvate is reduced to glycerate. Finally, glycerate is phosphorylated in the chloroplast to form 3-PGA, which can be fed back to the Calvin cycle [79,80].
The production of ROS is also an unavoidable consequence of the aerobic respiration. It occurs under normal respiratory conditions but can be enhanced in response to biotic and abiotic stress. ROS produced in mitochondria are regarded to be essential in PCD regulation . In mitochondria, O2•− is mainly produced in complex I, ubiquinone and complex III of ETC . This O2•− can be further converted into highly toxic •OH which may penetrate membranes and leave the mitochondrion . Hydroxyl radical can also initiate the peroxidation of mitochondrial membrane polyunsaturated fatty acids (PUFA) that leads to the formation of cytotoxic lipid aldehydes, alkenals and hydroxyalkenals, such as malonyldialdehyde (MDA). Lipid peroxidation products may cause cellular damage by reacting with other lipids, proteins and nucleic acids. The mitochondrial ETC produces significant amount of ROS but the mitochondrial enzyme - alternative oxidase (AOX) can prevent ROS overproduction . Some studies, performed on tobacco plants, have demonstrated that the lack of AOX induces PCD while the AOX overexpression decreases the lesion size during HR [85,86].
Another source of ROS - peroxisomes are small, spherical organelles with an oxidative type of metabolism. There are two sites of O2•− generation in peroxisomes: in the organelle matrix, where xanthine oxidase (XOD) catalyzes the oxidation of xanthine and hypoxanthine to uric acid and in the membrane, by components of peroxisomal ETC. The main metabolic processes responsible for H2O2 generation in peroxisomes are photorespiratory glycolate oxidase reaction, fatty acid β-oxidation, enzymatic reaction of ﬂavin oxidases and disproportionation of superoxide radicals .
ROS are also generated in the apoplast by NADPH oxidases residing in the plasma membrane and generating superoxide radicals. The extracellular O2•− is quickly mutated into H2O2 or converted to •OH. The latter initiates a series of reactions that cause a plasma membrane damage, finally leading to cell death. Two
The peroxidation of lipids is considered as one of the most damaging processes occurring in the cell. The damage of membrane is often considered as a parameter determining the level of cell destruction under various stresses. Upon ROS overproduction, polyunsaturated precursors undergo lipid peroxidation, forming small hydrocarbon fragments such as ketones or aldehydes. LPO in both cellular and organellar membranes affects proper cellular functions and aggravates oxidative stress by the production of lipid-derived radicals . This process often affects PUFA, since they contain multiple double bonds in between which lie methylene (-CH2-) groups with reactive hydrogens. Hydroxyl or perhydroxyl radicals combining with a hydrogen atom produce water and a fatty acid radical. The fatty acid radicals are unstable and react rapidly with molecular oxygen, creating a peroxyl-fatty acid radical (ROO•). Once initiated, ROO• can further propagate the peroxidation chain reaction by abstracting a hydrogen atom from PUFA side chains. The resulting lipid hydroperoxide easily decomposes into several reactive species including: lipid alkoxyl radicals, MDA, alkanes and lipid epoxides. Thus, LPO generates multiple peroxide molecules and results in the membrane ﬂuidity decrease, its leakiness to substances that do not normally cross it, the damage of membrane proteins and ion channels. It has been found that such PUFAs as linoleic and linolenic acids are particularly susceptible to ROS attack . Increased level of LPO has been demonstrated in many abiotic stress studies, for instance under salt stress in
Apart from lipid peroxidation, the accumulation of ROS leads to protein oxidation. Only few types of these covalent modifications are reversible, most of them are irreversible . A widely used marker for protein oxidation is their carbonylation level. The oxidation of amino acids such as arginine, histidine, lysine, proline, threonine and tryptophan causes the formation of free carbonyl groups, that may inhibit or alter the protein activity and increase the susceptibility towards proteolytic attack . Proteins with sulfur-containing amino acids and thiol groups are often the target for ROS. Cysteine and methionine are especially reactive with 1O2 and •OH. Activated oxygen radical can abstract the hydrogen atom from cysteine residue to form a thiyl radical that cross-links to a second thiyl radical and leads to the formation of disulphide bridges. Oxygen can also be added onto the methionine residue to form a methionine sulphoxide. The best characterized response to the oxidation of peptide residues is the induction of proteases that break down the oxidized proteins .
DNA damage, triggered by ROS is particularly dangerous for the cell since it causes replication errors and genomic instability. From all ROS, •OH has the most damaging effect to DNA as it can modify all components of the nucleic acid molecule: purines, pyrimidines and the deoxyribose backbone . The major types of DNA damage resulted from oxidative stress are the formation of dimers between adjacent pyrimidines, cross-links, base deletion, strand breaks and base modifications such as alkylation and oxidation. To counteract the DNA damage, plant cells evolved mechanisms for the DNA repair in both nucleus and mitochondria. These include the direct inversion of modifications or the replacement of the whole nucleotide .
To protect themselves against toxic oxygen intermediates, plant cells possess a vast antioxidant system. Stress-induced ROS accumulation is counteracted by both enzymatic and non-enzymatic antioxidants. Enzymatic ROS scavengers include superoxide dismutases (SOD), catalases (CAT), ascorbate peroxidases (APX), glutathione reductases (GR), monodehydroascorbate reductases (MDHAR), dehydroascorbate reductases (DHAR), glutathione peroxidases (GPX) and glutathione-S- transferases (GST). Low-molecular, non-enzymatic antioxidants include ascorbic acid (AsA), glutathione (GSH), proline, α-tocopherol, carotenoids and ﬂavonoids .
Metalloenzyme SOD is the most effective enzymatic antioxidant which is ubiquitous in all subcellular compartments. SODs remove O2•− by catalyzing its dismutation (Figure 4A):
O2•− + O2•− + 2H+ → 2H2O2 + O2
This reaction eliminates O2•− and hence decreases the risk of •OH formation. SODs are classified into three types, depending on their metal cofactor: copper/zinc (Cu/Zn-SOD), manganese (Mn-SOD) and iron (Fe-SOD). Different types of SODs are located in different cellular compartments .
Catalases are tetrameric enzymes containing heme with the potential to dismutate H2O2 into H2O and O2.
2H2O2 → 2H2O + O2
CAT1 and CAT2 are localized in peroxisomes and cytosol, whereas CAT3 is targeted to mitochondria. Increased CAT activity has been reported in various abiotic stress studies in different species, e.g. under drought stress in wheat . Moreover, a vast number of research indicate that CAT overexpression leads to the abiotic stress tolerance, e.g. wheat catalase expressed in transgenic rice has been demonstrated to improve the tolerance against low temperatures .
Another group of antioxidising enzymes - ascorbate peroxidases are involved in H2O2 scavenging in water-water and glutathione-ascorbate cycles and use ascorbic acid as the electron donor (Figure 4A and B). The reaction catalysed by APXs is the transfer of electrons from ascorbate to hydrogen peroxide, producing dehydroascorbate and water
H2O2 + C6H8O6 → 2H2O + C6H6O6
Glutathione reductases are oxidoreductases participating in the glutathione-ascorbate cycle (Figure 4B). They play an essential role in the defense against ROS by sustaining reduced status of glutathione (GSH), a tripeptide molecule involved in many regulatory and antioxidative processes in plants. They are localized predominantly in chloroplasts, but small amounts have been also found in mitochondria and cytosol . GRs catalyze the NADPH-dependent reduction of the oxidized form of glutathione (GSSG) (Figure 4B and C) thus are important for maintaining the GSH pool. Increased GR activity has been demonstrated in various abiotic stress studies, e.g. in drought stressed rice seedlings . Transgenic plants with lower GR activity have shown enhanced sensitivity to oxidative stress while these with higher GR have been proved to be abiotic stress tolerant. Elevated chloroplastic GR activity has been demonstrated to decrease chilling-induced photoinhibition in transgenic cotton .
Monodehydroascorbate reductase is an enzymatic component of the glutathione-ascorbate cycle (Figure 4B). MDHARs are present in chloroplasts, mitochondria, peroxisomes and cytosol, where they participate in H2O2 scavanging . They exhibit high specificity for monodehydroasorbate as the electron acceptor and use NADH as the electron donor (Figure 4B):
NADH + C6H7O6 → NAD+ + C6H8O6
Overexpression of MDHAR in the transgenic tobacco has been demonstrated to increase the tolerance against ozone, salt and osmotic stress .
Dehydroascorbate reductases function in the regeneration of ascorbic acid from the oxidized form (Figure 4B) and therefore regulate cellular AsA redox state. DHAR overexpression has been demonstrated to enhance salt tolerance in
Glutathione S-transferases are a large and diverse group of enzymes with 54 members reported in
Glutathione peroxidases are another group of isoenzymes that use GSH to reduce H2O2, organic and lipid hydroperoxides (Figure 4C). A family of seven related proteins (AtGPX1-AtGPX7) residing in cytosol, chloroplasts, mitochondria and endoplasmic reticulum has been identified in
Apart from enzymes participating in redox homeostasis maintenance, plants possess a vast number of non-enzymatic compounds acting as antioxidants. Ascorbic acid (vitamin C) is present in all plant tissues but especially high levels occur in photosynthetically active organs, meristems and some fruits. Mitochondria play a central role in the metabolism of AsA in plants. Ascorbic acid reacts with oxidants such as O2•− and •OH, transfering a single electron and forming its own radical ion in the following reaction:
RO• + C6H7O6− → ROH + C6H6O6•−
The oxidized form of ascorbate - dehydroascorbate (DHA) is relatively unreactive and do not cause cellular damage. However, it has a short lifetime and needs to be regenerated back into AsA. Ascorbic acid antioxidative capacity provides a protection to membranes by direct scavenging ROS and by regenerating α-tocopherol from tocopheroxyl radical. In chloroplasts, AsA acts also as a violaxantin de-epoxidase cofactor, sustaining dissipation of excess excitation energy .
Another powerful antioxidant – glutathione is a tripeptide (γ-glu-cys-gly). In plant tissues it occurs in a reduced form (GSH) and plays a central role in several physiological processes, including detoxiﬁcation of xenobiotics, signal transduction, conjugation of different metabolites, differentiation, senescence and cell death regulation . By serving as an electron donor, GSH is converted into oxidized form - two glutathione molecules linked by a disulﬁde bond (glutathione disulfide, GSSG). Once oxidized, glutathione can be reduced by glutathione reductases that use NADPH as an electron donor (Figure 4C). The GSH/GSSG ratio is often used as a measure of cellular redox state. GSH is necessary to maintain reduced state of cell, counteracting inhibitory effects of ROS. It plays a key role in the antioxidative defense system by regenerating other antioxidants like AsA via the glutathione-ascorbate cycle (Figure 4B). GSH is particularly important in chloroplasts since it helps to protect the photosynthetic apparatus from oxidative damage .
Proline is considered as another important antioxidant and potential inhibitor of PCD. It has been well established that it acts as an osmoprotectant and protein-stabilizing agent. However, it has been also proven to be the O2•− and •OH scavenger and inhibitor of LPO . Increased concentration of proline has been correlated with enhanced tolerance to various abiotic stresses, e.g. transgenic tobacco cells with silenced proline dehydrogenase, accumulating more proline than wild-type cells, have shown improved osmotolerance . Over-expression of proline biosynthetic pathway genes has been also found to increase the drought stress tolerance in transgenic soybean .
Out of four tocopherol isomers (α, β, γ, δ) found in plants, α–tocopherol (vitamin E) has the highest antioxidative activity because of the presence of three methyl groups . α-tocopherol, a lipid soluble antioxidant molecule is considered as a potential scavenger of ROS and lipid radicals in membranes. It has been shown to prevent the chain propagation step in the lipid autooxidation reaction . It has been demonstrated that oxidative stress activates the expression of tocopherols synthesis pathway genes. Higher tocopherol level has also been reported during water stress .
Another group of plant compounds with antioxidant abilities are lipid soluble carotenoids. They play various functions in the plant metabolism such as absorption of light at wavelength between 400 and 550 nm (light-harvesting role), assembly and stabilization of light harvesting complex proteins (structural role), and protection of the photosynthetic apparatus from free radicals (antioxidant role) .
Apart from the antioxidant function, flavonoids are responsible for ﬂowers, fruits and seeds pigmentation, protection against UV, defense against pathogens and signal transduction during stress. Mutant plants, defﬁcient in chalcone synthase and chalcone isomerase that are unable to accumulate ﬂavonoids have been demonstrated to be more sensitive to UV light . Many genes encoding ﬂavonoid biosynthesis components are induced under stress conditions. Considerable increase in ﬂavonoid level has been demonstrated in response to abiotic stresses such as wounding, drought and nutrient deprivation .
Under steady state conditions, ROS are eliminated by antioxidative mechanisms described above (78). Different abiotic and biotic stresses such as drought, high salinity, heavy metals, high light, UV radiation, high/low temperature or pathogen attack may disturb the balance between the ROS production and scavenging. The equilibrium between ROS production and scavenging influences their mode of action as protective, signaling or damaging factors. The increase in cellular ROS level can cause significant damage to cell structures, cell death and in consequence loss in crop production . The vast role of ROS in the response to environmental conditions and cell-death signaling are well documented [65,128]. There are results suggesting that H2O2 antagonizes the 1O2-mediated signaling and that the cross-talk between signaling pathways, transferred by different ROS, may contribute to the overall response of plant exposed to adverse environmental conditions . Moreover, ROS interact with several other signaling pathways including NO and hormones like SA, JA and ET. Such interactions and the ROS/hormonal balance determine whether the cell will stay alive or enter the PCD pathway [14,39,60]. Finally, the role of ROS as messenger molecules cannot be underestimated, since it has been demonstrated that they trigger the transduction of stress signals and systemic acclimation to adverse environmental conditions [130,131].
4. High and excess light stress
Light is an essential factor in the regulation of plant growth, development and stress responses but it is also responsible for the production of reactive oxygen species leading to PCD. The cell death phenotype of many lesion mimic mutants of
In natural environment, plants are often exposed to high light (HL) intensities that lead to the absorption of more light energy than can be used for carbon dioxide ﬁxation . The amount of absorbed light energy that is excessive and cannot be used for photosynthetic metabolism is termed excess excitation energy (EEE) [77,130]. In response to EEE, there is an immediate increase in the electron transport rate and in consequence redox changes of PET components. Alterations in the redox status of PET, especially the reduction of PQ pool leads to the expression deregulation of nuclear and chloroplastic genes that encode photosynthesis components such as LHC proteins [141–143] and antioxidants like APX [144,145]. The response to EEE involves not only the alteration in photosynthetic ﬂux but it is also accompanied by changes in the water status and temperature of the leaf, and in consequence it is associated with elevated ABA levels, changes in the redox state of glutathione pool and increased activity of heat shock transcription factors [146,147]. If the accumulation of ROS exceeds the ability of removing them by antioxidant systems, it may cause a photooxidative damage of the photosynthetic apparatus which may lead to cell death, manifested by bleaching, chlorosis or bronzing of leaves . Therefore, the avoidance of EEE, its dissipation and HL tolerance are fundamental for the plant survival. EEE-mediated PCD can be considered as a beneficial process, as it triggers signal transduction to systemic cells and their acclimation to high light [130,131].
Avoidance strategies include such processes as: movements of chloroplasts, decrease in the number of photosynthetic reaction centers, curling of leaves and increase in the thickness of cuticular wax . During HL treatment, chloroplasts have been demonstrated to move to the anticlinal wall (Figure 5) and this response has been proven to be mediated by blue/UVA receptors .
Plants have also developed several mechanisms for removing the excess of energy. Dissipation of EEE can be achieved by the combination of photochemical (qP) and non-photochemical quenching (NPQ) processes. Photochemical quenching increases the utilization of photosynthetic electrons by metabolic pathways such as the water-water cycle or photorespiration. The consumption of electrons through water-water cycle is achieved by combined action of the O2 reduction at PSI to O2− and the chloroplast antioxidant system. The reduction of O2 is much lower than the disproportionation of O2−, catalysed by SOD and the following H2O2 processing to H2O, catalysed by APX. Therefore, the water-water cycle shortens the lifetime of O2− and H2O2, suppresses the production of •OH, and prevents photoinhibition . Another energy sink preventing photoinhibition of photosynthetic apparatus by EEE is photorespiration. During this process, photo-produced ATP and reducing equivalents are consumed, preventing the overreduction of PET. However, the photorespiratory cycle leads to the production of H2O2 that has to be elimainated by antioxidant systems .
Non-photochemical quenching processes relay on the transfer of excitation energy to carotenoids that are able to dissipate it as heat during the xanthophyll cycle (VAZ cycle). The xanthophyll cycle involves the conversion of violaxanthin to de-epoxidised zeaxanthin, via the intermediate antheraxanthin. This enzymatic cycle is performed by violaxanthin de-epoxidase and plays a key role in the stimulation of energy dissipation within light-harvesting antenna . In
Excess energy is sensed by the photosynthetic apparatus not only as a result of HL, but also other environmental factors such as UV radiation, limitations in nutrient availability, drought, salinity or high/low temperatures. All these abiotic stimuli are accompanied by oxidative stress, manifested in the overproduction of reactive oxygen species. If the level of ROS is too high for antioxidant system to eliminate them, cellular macromolecules and structures can be damaged, which triggers PCD. Several studies clearly demonstrate that programmed cell death is affected by light. The spread of wound-induced PCD in maize tissue has been shown to be transmitted by chloroplast-produced ROS . It has also been found that the HR-mediated cell death is accelerated by the loss of chloroplast function . Moreover, a study using light- and dark-grown plant cell culture has proven that they respond differently to PCD-inducing stimuli, resulting in various levels of DNA fragmentation and cell-content condensation . Direct induction of programmed cell death by exposure of
Although ROS produced during the progress of PCD are damaging for the cell, they are also needed as messenger molecules preparing other cells for a struggle with stress conditions. H2O2 is thought to freely diffuse across biological membranes, thus it has been proposed to directly influence the function of extra-chloroplastic signaling components. The possibility that H2O2 acts as an intracellular messenger molecule has been suggested since it triggeres systemic response to EL . When low-light-grown
In the last decade a significant progress has been made in improving light-induced oxidative stress tolerance in plants. Various components of antioxidative system involved in ROS scavenging have been up- or down-regulated to develop transgenic lines with altered antioxidants levels. Overexpression of enzymes involved in AsA biosynthesis has been shown to confer oxidative stress tolerance in tomato plants . Increased AsA content has been also demonstrated to enhance high light stress tolerance in
5. UV radiation stress
Being exposed to sunlight, plants need to deal with the damaging effect of ultraviolet (UV) radiation which reduces the genome stability, impeding their growth and productivity. These effects result from damage to cell components including not only nucleic acids, but also proteins and membrane lipids. Upon UV exposure, strongly mutagenic cross-linked forms of DNA can be produced . In order to minimize effects of UV radiation, plants accumulate UV-absorbing secondary metabolites, perform the monomerization of UV-induced pyrimidine dimers (DNA repair) and neutralize generated ROS [166,167].
UV radiation consists of UV-C (below 280 nm), UV-B (280-320 nm) and UV-A (320-390). Although UV-C is not physiologically relevant to plants since it is efficiently blocked by the stratosphere, the UV-C-triggered cell damage is comparable to induced with UV-B radiation, which reaches Earth's surface . Therefore, UV-C radiation has been widely used to study DNA damage and repair mechanisms upon UV stress .
UV has been demonstrated to trigger apoptosis in animals  and apoptosis-like changes in
6. Drought stress - soil water deficit
Drought is one of the most unfavorable environmental factors that affects growth and development of plants and consequently limits plant productivity. Plants have developed specific acclimation and adaptation mechanisms to survive the soil water deficit. In response to drought, plants can exhibit either escape (ability to complete the life cycle before severe stress) or resistance mechanisms. Resistance mechanisms include drought avoidance and drought tolerance. The latter depends on the cell turgor maintenance by accumulating osmolytes and soluble sugars . There are several examples of molecules that help to maintain an osmotic balance under dehydration conditions: sugars, polyols and proline . Proline is accumulated in the cytoplasm and chloroplast stroma while other solutes (sugars, organic acids, potassium) are cumulated in the vacuole. When the cellular water content decreases, they stabilize cellular structures through hydrophilic interactions and hydrogen bonding . A similar role is fulfilled by late embryogenesis abundant (LEA) proteins - a family of unstructured proteins. LEA proteins accumulate in response to dehydration and ABA treatment. Because of their high hydrophilicity and solubility in water, it has been proposed that they play a role in protecting cytoplasmic structures during dehydration . The avoidance mechanism is possible by the maintenance of high water potential in plant tissue despite soil water deficit. It can be achieved by: improved water uptake under stress, the ability to hold water as well as by the reduction of its loss through smaller leaf area and lesser stomatal and cuticular conductance. One of the first acclimation responses to drought is the decrease in leaf growth, which helps to maintain the cell turgor and reduces the transpiration area. In
Photosynthesis is one of major processes affected by water deficit since stomata closure causes reduced CO2 diffusion to the chloroplast. As a result of the inhibition of photosynthesis and the predominance of photorespiration, ROS are generated . It has been demonstrated that in drought-stressed plants, the ABA-controlled stomata closure is mediated by H2O2 . Under severe drought stress, some antioxidant enzymes have been shown to be highly induced . However, studies on many drought-stressed crop species showed an inconsistency in their expression since in some cases they have been induced, but in other repressed, suggesting that different ROS balance may be required during different response phases .
During water deficit, ROS are responsible for the induction of leaf senescence, which is executed through the programmed cell death and plays an important role in the plant survival. It contributes to the nutrients remobilisation during stress and allows the rest of plant to benefit from them and stay alive. Drought-induced PCD enables also the abscission of some leaves and thus the avoidance of further water loss through the transpiration. It occurs gradually and is manifested by specific biochemical and molecular changes such as chromatin condensation, thylakoid swelling, lipid peroxidation, degradation of chlorophyll (leaf yellowing) and proteins. Apart from ROS, cytokinins and ABA have been shown to be involved in the regulation of water-deficit-triggered senescence . Recent studies have shown that the water deficit triggers PCD not only in green tissues but also in plant root tips. Apical meristem cells of primary roots undergoing PCD, demonstrate increased size of vacuole, degradation of organelles and the collapse of plasma membrane .
Early events in the perception of drought stress signals include the activation of transcription factors belonging to such classes as DREB/CBF (e.g. DREB1a, DREB2a), ABF (e.g. ABF2, ABF4), MYB (e.g. MYB2), MYC, NAC and WRKY. Many of them possess stress responsive cis-regulatory elements in their promoter sequences like abscisic acid-responsive elements (ABRE) and drought-responsive elements (DRE) [189–191]. The plasma membrane-associated NTL4 (NAC transcription factor) after drought or ABA treatment has been shown to be proteolytically activated and transported to the nucleus where it induces expression of NADPH oxidase involved in ROS generation . Moreover, the dehydration stimulates expression of BAX inhibitor-1 (AtBI-1). The
Upon soil water deficit, the accumulation of ABA and the induction of ABA-associated signaling genes occur. ABA induces various second messengers such as cytosolic Ca2+, ROS and NO in guard cells. These signals evoke ion efflux through plasma membrane ion channels, resulting in the reduction of guard cell turgor pressure and stomata closure to reduce water loss through the transpiration . Mutants with the perturbation of ABA synthesis or signaling display drought hypersensitivity, manifested in significant growth reduction which suggests that ABA is needed for the proper response to drought . In
Similarly to ABA, JA also triggers stomata closure and such response is conserved among various plant species . At the early stage of moderate drought, plants accumulate high concentrations of ABA and induce ABA-responsive genes. At this stage, no significant differences in JA-responsive genes are observed. At later stage of drought stress, ABA level returns to normal, while JA synthesis and JA signaling genes are significantly down-regulated. This suggests the negative correlation between ABA and JA pathways . The high concentration of JA is probably undesirable during drought stress, as it inhibits the cell expansion and results in stunted growth . Therefore, plants down-regulate JA synthesis and signaling pathways to minimize the inhibitory effect of JA on growth, establishing a new hormone homeostasis.
Downstream of early stress perception events, signaling molecules are activated. Such secondary messengers include Ca2+ ions and ROS. They induce further genes that are needed to establish a new cellular homeostasis leading to drought resistance and tolerance . Recent studies have strongly proven that drought response progresses through mitogen-activated protein kinase (MAPK) pathways . In yeast and animals, MAPK-regulated pathways take part in the production of osmolytes and antioxidants. These MAPK pathways are activated by receptors/sensors such as protein tyrosine kinases, G-protein–coupled receptors and histidine kinases. Among these, G-protein–associated receptors have been proposed to serve as one kind of membrane-bounded receptors for ABA. A family of histidine kinases (HK) have been also identified in plants . An
The elucidation of mechanisms controlling drought stress responses has enabled to engineer plants by the expression of specific stress-related genes. Although it was believed that the modulation of osmoregulatory genes would be the best strategy, attempts failed to result in any significant drought-stress tolerance improvement . However, constitutive expression of some LEA proteins has conferred tolerance to soil water deficit in transgenic rice  and wheat . Moreover, tomato plants overexpressing
Plants have evolved various strategies to acclimate to different environmental stresses. The most fundamental strategy is the development of high plasticity of plant tissues. It has been demonstrated that programmed cell death plays an important role in this plasticity and subsequent adaptation to unfavorable conditions. There is a growing evidence that PCD is a crucial process in both morphogenetic changes execution and the following adaptation. Although each decade brings a vast number of research, our understanding of plant PCD and its underlying mechanisms is still in its early stage. Further insight into details of the PCD molecular machinery in plants is important, since it is an attractive target for improving stress tolerance and plant yield under adverse conditions. Essentially, it could lead to the generation of pathogen-resistant and stress-tolerant crops as well as fruit varieties with an extended shelf life.
This work was supported by the Welcome/2008/1 Program operated within the framework of the Foundation for Polish Science, co-financed by the European Regional Development Fund.
ABA - abscisic acid; AOX - alternative oxidase; APX - ascorbate peroxidase; AsA - ascorbic acid; BI - Bax-Inhibitor; CAT – catalase; Chl – chlorophyll; DBMIB - 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; DCMU - 3,4-dichlorophenyl-1,1-dimethylurea; DHA – dehydroascorbate; DHAR - dehydroascorbate reductase; EEE - excess excitation energy; EL - excess light; ER - endoplasmic reticulum; ET – ethylene; ETC - electron transport chain; FAD - flavin adenine dinucleotide; GA - gibberellic acid; GPX - glutathione peroxidase; GR - glutathione reductase; GSH - reduced glutathione; GSSG – oxidized glutathione; GST - glutathione-S- transferase; HL - high light; IAP - inhibitor of apoptosis; JA - jasmonic acid; HR - hypersensitive response; LEA - late embryogenesis abundant; LHC - light-harvesting complex; LPO - lipid peroxidation; LSD1 - LESION SIMULATING DISEASE 1; MC – metacaspase; MDA – malonyldialdehyde; MDHAR - monodehydroascorbate reductase; MeJA - methyl-jasmonic acid; MAPK - mitogen-activated protein kinase; NADPH - nicotinamide adenine dinucleotide phosphate; NO - nitric oxide; NPQ - non-photochemical quenching; PA - phosphatidic acid; PCD - programmed cell death; PEPS - photoelectrophysiological signaling; PET - photosynthetic electron transport; PG – phosphoglycolate; PGA – phosphoglycerate; PLD - phospholipase D; PSI and PSII - photosystem I and II; PQ – plastoquinone; PUFA - polyunsaturated fatty acids; qP - photochemical quenching; RCD - runaway cell death; RNS - reactive nitrogen species; ROS - reactive oxygen species; Rubisco - ribulose-1,5-bisphosphate carboxylase/oxygenase; RuBP - ribulose-1,5-bisphosphate; SA - salicylic acid; SAA - systemic acquired acclimation; SAR - systemic acquired resistance; SOD - superoxide dismutase; TF - transcription factor; UV - ultraviolet radiation; VPE - vacuolar processing enzyme; XOD - xanthine oxidase.