Novel insight into functions of ascorbate peroxidase in higher plants: More than a simple antioxidant enzyme

Abstract

As plants are sessile organisms, they are inevitably exposed to a variety of environmental stimuli that trigger rapid changes in the generation and disposal of reactive oxygen species such as hydrogen peroxide (H₂O₂). A major H₂O₂ scavenging system in plant cells is the ascorbate-glutathione cycle, in which ascorbate peroxidase (APX) catalyzes the conversion of H₂O₂ into water employing ascorbate as specific electron donor. In higher plants, distinct APX isoforms can occur in multiple subcellular compartments, including chloroplasts, mitochondria, and peroxisomes and the cytosol, to modulate organellar and cellular levels of H₂O₂. It is well established that APX plays crucial roles in protecting plant cells against diverse environmental stresses, as well as in plant growth and development. Apart from ascorbate, recently, APXs have been found to have a broader substrate specificity and possess chaperone activity, hence participating various biological processes. In this review, we describe the antioxidant properties of APXs and highlight their novel roles beyond ‘ascorbate peroxidases’.

1. Introduction

Reactive oxygen species (ROS), such as, singlet oxygen, superoxide anion, hydroxyl radical, and hydrogen peroxide (H₂O₂), are inevitable components of aerobic metabolism in all living organisms [1]. When plants are exposed to harsh environmental conditions, ROS levels can increase excessively and cause marked oxidative damage to DNA, RNA, proteins, lipids, and other redox-sensitive molecules. Therefore, plants must develop a sophisticated antioxidant system to modulate cellular ROS concentrations [[2], [3], [4]]. Among the ROS compounds, H₂O₂ is fairly stable and can transport between cellular compartments through aquaporins, which function as a cellular signaling molecule to myriad of biological processes such as stress responses, growth and development [5,6]. Accumulating evidence suggests that H₂O₂ largely signals via oxidative post-translational modifications (OxiPTMs) of proteins, enabling proteins to fine-tune their conformational states and activities [1,7].

In plants, H₂O₂ can be scavenged by several antioxidant enzymes, including catalases (CATs), ascorbate peroxidases (APXs), peroxiredoxins (PRXs) and glutathione S-transferases (GSTs) with different mechanisms [8]. Among them, the heme-containing CAT and APX are two key enzymes associated with H₂O₂ metabolism. CATs are mainly localized in peroxisomes and degrade H₂O₂ without reductants. APX has a higher affinity for H₂O₂ than CAT and catalyzes the reduction of H₂O₂ to water using ascorbate (ASC) as an electron donor in various subcellular compartments (Fig. 1) [[9], [10], [11], [12]]. In Arabidopsis thaliana, eight AtAPX genes were reported previously, which encode three cytosolic (cytAPXs: AtAPX1, 2, and 6), three peroxisomal (perAPXs: AtAPX3, 4 and 5), and two chloroplastic (chlAPXs: soluble stromal AtsAPX, and thylakoid membrane-bound AttAPX) isoforms [13]. However, AtAPX4 and AtAPX6 are unlikely to encode classical APXs. Due to lack of essential catalytic residues, ASC-binding and heme-binding sites [13], AtAPX4 (also termed TL29) has been renamed APX-like (APX-L) [14]. However, the Arabidopsis apx4 null mutants exhibit decreased soluble APX activity and increased H₂O₂ accumulation, suggesting a H₂O₂-scavenging role of APX-L [15]. AtAPX6 is a heme peroxidase that does not utilize ASC as substrate to reduce H₂O₂ [16], and has been considered to belong to APX-related (APX-R) family [16,17]. Moreover, AtAPX6 was demonstrated as a chloroplast targeted protein [16], though it was previously reported as a cytosolic protein. In recent years, APX-L and APX-R have been reclassified as two novel families of class I peroxidases [14]. Therefore, Arabidopsis harbors only six functional APXs. In contrast to APXs encoded by multiple genes [18,19], both APX-L and APX-R are generally encoded by a single gene in plants [20].

Fig. 1.

Main sources of ROS formation in the plant cell. ¹O₂ is mostly produced at PSII in thylakoid membranes of chloroplasts whereas both O₂^•− and H₂O₂ are generated at several subcellular and extracellular sites. The O₂^•− is converted to H₂O₂ either via spontaneous dismutation or by the action of SODs. The levels of H₂O₂ are controlled by ASC-GSH cycle, which operates in various cellular compartments, including the cytosol, chloroplasts, mitochondria and peroxisomes [82,83]. H₂O₂ might cross biological membranes via aquaporins. In the reaction with H₂O₂, ASC is oxidized to MDHA, which can rapidly disproportionate to produce DHA and ASC. Using GSH as the reducing agent, DHA is reduced to ASC either chemically or via the action of DHAR. Abbreviations: ASC, ascorbate; APX, ascorbate peroxidase; CAT, catalase; DHA(R), dehydroascorbate (reductase); GOX, glycolate oxidase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, glutathione disulphide; MDHA(R), monodehydroascorbate (reductase); 2 PG, 2-phosphoglycolate; PGA, 3-phosphoglycerate; PSI/II, photosystem I/II, RETC, respiratory electron transport chain; RBOH, respiratory burst oxidase homolog; RuBisCO, ribulose 1,5-bisphosphate carboxylase/oxygenase; RuBP, ribulose 1,5-bisphosphate; SOD, superoxide dismutase; XO, xanthine oxidase.

As an essential enzyme of the ascorbate-glutathione (ASC-GSH) cycle [21], APX has been showed to participate in many plant growth and development processes, such as lateral root formation [22], nodule development [23], leaf senescence [24], seed germination [25], and programmed cell death (PCD) [26], and play a pivotal role in plant responses to environmental stimuli that have been extensively reviewed previously [18,27]. For example, ZmAPX1-overexpressing transgenic maize plants display enhanced resistance against Bipolaris maydis by decreasing H₂O₂ levels and activating the jasmonic acid signaling pathway [28]. Arabidopsis plants overexpressing AttAPX exhibit enhanced resistance to paraquat-induced stress [29]. Often, stacking of APX and other antioxidant enzymes makes transgenic plants more tolerant to various stresses [[30], [31], [32]]. Although these data indicate the importance of APX in maintaining the H₂O₂ homeostasis under adverse environmental conditions, its functional role may be more complicated. For instance, Arabidopsis apx1 mutants showed enhanced tolerance to selenium and lead [33,34]. Arabidopsis mutants deficient in APX2 exhibited reduced tolerance to heat stress during the seedling stage, but enhanced thermotolerance during the reproductive stage [35]. Simultaneous deficiency of APX and CAT are more resistant to oxidative stress than the respective single mutants [[36], [37], [38]].

Our following discussion is not to repeat the importance of APX in plant stress response, growth and development, but rather focus on recent advances, highlighting the OxiPTMs of APX, and new facets of APX including their, newly-discovered substrates in vivo, and chaperone activity.

1.1. OxiPTMs of cytAPX

Although APX is the key regulator in sustaining the steady-state levels of H₂O₂ in plant cells, it is subjected to multiple OxiPTMs (Fig. 2), and the cytAPX is the best-studied isoform. The cytAPX was identified as a target of thioredoxins (TRXs) by proteomic approaches [[39], [40], [41]]. Treatment of recombinant cytAPX with TRX or GSH inhibited its activity dramatically, which suggests that activation of cytAPX is associated with cysteine (Cys) oxidation or glutathionylation [42]. The Cys thiols (-SH) are particularly susceptible to oxidation by H₂O₂, and their reaction leads to the formation of sulfenic acid (-SOH, sulfenylation). In addition to H₂O₂, Cys thiols of APX can undergo a range of other OxiPTMs. Nitric oxide (NO) and hydrogen sulfide (H₂S), the most studied reactive nitrogen species (RNS) and reactive sulfur species (RSS), can react with Cys thiols to generate S-nitrosated and persulfidated residues, respectively [43]. The S-nitrosation of Arabidopsis APX1 at Cys32 elevates its enzymatic activity of removing H₂O₂, leading to enhanced resistance to oxidative stress [44]. By contrast, a rapid reduction in cytAPX activity due to S-nitrosation has also been reported in tobacco cells during PCD [26]. Persulfidation by H₂S has been observed to occur at Cys residue that stimulates cytAPX activity in both Arabidopsis and tomato [45,46]. Furthermore, Cys thiols of APX also can react with other cellular compounds such as fatty acids (FAs) or cyanide (HCN), referred to as S-acylation or S-cyanylation [47,48], and their effects on cytAPX need to be explored. During conditions of strong oxidative stress, competing OxiPTMs might take place at Cys residues simultaneously, which makes the modifications of APX more complicated to be analyzed though exciting.

Fig. 2.

Main OxiPTMs of APX in plants. The main PTMs that can affect the of Cys thiols of APX, including S-sulfenylation, persulfidation, S-nitrosation, S-glutathionylation, S-cyanylation and S-acylation. OxiPTMs also occur at Met and Tyr residues of APX. Furthermore, actitity of APX can be inhibited by the reversible binding of NO to the heme prosthetic group. Besides specific sites, H₂O₂ can attact romdom sites of APX, such as carboxylation. Solid arrows denote activation, whereas the T-shaped lines indicate inhibition. The solid arrow and question mark symbolize contradictory data about the way that S-nitrosation affects APX activity. Dash arrows represent the regulatory effect remains to be elucidated.

Similar to Cys, the sulfur-containing methionine (Met) residues are susceptible to H₂O₂. In banana, Met oxidation (sulfoxidation) in cytAPX inactivates its activity, which can be reversed partially by Met sulfoxide reductase B2 [49]. Moreover, RNS have been described to regulate APX function through tyrosine (Tyr) nitration and metal nitrosylation. In citrus plants exposed to salinity stress, cytAPX gets nitrated in roots but not in shoots [50]. Interestingly, cytAPX from pea is modulated by RNS in a dual fashion. While nitration at Tyr5 and Tyr235 residues by peroxynitrate (ONOO⁻) inhibits APX activity, S-nitrosation at Cys32 residue enhances its activity [51]. In tobacco, NO interacts with the iron atom of the heme prosthetic group of APX, leading to reversible inhibition of enzyme activity [52].

Apart from specific site modifications (e.g., Cys and Met oxidation), OxiPTMs also occur at more random sites. Carbonylation at arginine (Arg), lysine (Lys), proline (Pro) or threonine (Thr) residues irreversibly inhibits APX activity, during seed dehydration of Antiaris toxicaria [53].

1.2. APX participates in lignin biosynthesis

Lignin is the second most plentiful biopolymers on earth after by cellulose, accounting for ca. 30% of the organic carbon in the biosphere [54]. It primarily consists of three monolignols, p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. Upon their production in the cytoplasm, monolignols are transported to the apoplastic space. Once secreted, monolignols are oxidized by class III peroxidases (utilizing H₂O₂) and/or laccases (using molecular oxygen) to form p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) units of lignin, respectively. In the lignin biosynthesis pathway, the conversion of coumarate to caffeate was previously proposed to be catalyzed by a complex of two membrane-bound cytochrome P450 enzymes, viz. the cinnamate 4-hydroxylase and coumaroyl shikimate 3′-hydroxylase [54]. Recently, a soluble coumarate 3-hydroxylase (C3H) has been identified as a bifunctional APX (C3H/APX) that can oxidize both ASC and 4-coumarate at comparable rates in the cytosol of Brachypodium distachyon and Arabidopsis [55] (Fig. 3). Structural modeling of the of C3H/APX showed that 4-coumarate and ASC bind at the δ- and γ-edges of the heme cofactor, respectively [55]. The cytAPX (SbAPX) from sorghum (Sorghum bicolor) was also demonstrated to catalyze the 3-hydroxylation of 4-coumarate in the presence of ASC and H₂O₂. However, most caffeate was produced from a nonenzymatic reaction and hence the C3H activity by SbAPX might be negligible in sorghum [19]. Therefore, to what extent the bio-functionality of APX/C3H is conserved among higher plants remains to be clarified.

Fig. 3.

Multiple substrates of APX identified in plants. In addition to ASC, APXs have been found to accept other substrates, such as glutathione, 4-coumarate and sinapyl alcohol in plants [56,60,84].

In addition, Zhang et al. (2022) has reported that mitochondrial APX (PtomitAPX) of Chinese white poplar (Populus tomentosa) detoxifies H₂O₂ through the ASC-GSH cycle in mitochondria in living cells, while in fibres and tracheary elements, PtomitAPX is translocated to cell walls during PCD and recruits monolignols as substrates to catalyze monolignol polymerization during the early stage of lignification. In the late stage of lignification, however, lignin polymers may be catalyzed by class III peroxidases and laccases rather than PtomtAPX [56]. Enzymatic activities tests showed that PtomtAPX could catalyze all three monolignols, with the fastest reaction rates for sinapyl alcohol, followed by p-coumaryl alcohol and coniferyl alcohol, however, the catalytic efficiency (K_cat/K_m) was pronouncedly lower for the monolignols than for ASC [56]. Similar to PtomtAPX, SbAPX was also suggested to participate in lignin polymerization because it could catalyze oxidative polymerization of multiple phenylpropanoid intermediates in the monolignol pathway [19]. Nevertheless, whether SbAPX is translocated to the cell wall was not conducted.

Furthermore, the availability of H₂O₂ is a restricting factor in lignification [57]. Simultaneous overexpression of APX and CuZn-superoxide dismutase in potato resulted in enhanced expression of genes and transcription factors associated with lignin biosynthesis under salinity stress [58]. It suggests that enhancement of APX activity plays a vital role in maintaining an optimal level of H₂O₂, which might serve as a signal to stimulate lignification under saline conditions. As far as the bifunctionality of APX is concerned, which activity is the causative factor in lignin synthesis under external stimuli remains to be explored.

1.3. GSH oxidation activity of APX

ASC and GSH often are considered to work together to scavenge ROS. In the ASC-GSH cycle, GSH can regenerate ASC by reducing dehydroascorbate (DHA), either via non-enzymic reduction or through DHA reductase (DHAR) activity [59]. Recently, Chin et al. (2019) has reported that a cytAPX of orchid Oncidium (OgAPX1) can utilize not only ASC but also GSH as a substrate at different active sites [60] (Fig. 3). Structural modeling and site-directed mutagenesis demonstrate that Pro63, aspartate (Asp)75 and Tyr97 are necessary for GSH oxidation in OgAPX1, whereas the corresponding site in AtAPX1 is comprised of Asp63, histidine (His)75 and His97 without GSH-binding activity. The dual catalytic activity of OgAPX1 confers greater protection against salt and heat tolerances when overexpressed in Arabidopsis compared with overexpression of AtAPX1. Aside from OgAPX1, recombinant cytAPXs from rice, maize and soybean also possess GSH oxidation activity that refers to glutathione peroxidase (GPX) [60].

It is worth noting that most plant GPXs preferentially utilize TRX rather than GSH as a reducing agent [[61], [62], [63]], and therefore sometimes are called GPX-like proteins [64]. In plants, the oxidation of GSH to glutathione disulfide (GSSG) can be also attributed to several other enzymes, including DHAR, lambda class of GSTs with peroxidase activity and certain PRXs that are coupled GSH oxidation via glutaredoxin (GRX) action [65]. Notably, Arabidopsis DHAR has been identified as a crucial player in ensuring GSH oxidation in response to intracellular oxidative stress [66,67]. To fully elucidate the GSH oxidation mechanism of OgAPX1, it is necessary to determine the substrate specificity of GPX in orchid Ocidium, and the dual catalytic activity of OgAPX1 would be redefined.

1.4. APX functions as a molecular chaperone

Besides their primary antioxidant properties, APXs have been identified as chaperone molecules in Arabidopsis (AtAPX1) [68] and rice (Oryza sativa, OsAPX2) [69]. The dual functions of AtAPX1 and OsAPX2 correlate closely with their structural conformations. The low-molecular-weight (LMW) forms of the AtAPX1 and OsAPX2 predominantly exhibit peroxidase activity, whereas the high-molecular-weight (HMW) complexes display chaperone activity. Moreover, structural status of the AtAPX1 and OsAPX2 proteins could be modulated by heat and salt stresses through association and disassociation, respectively [68,69]. Whether a peroxidase-chaperone functional switch of APX is conserved in land plants will require further exploration.

As with AtAPX1, 2-Cys PRX changes its structure from LMW to HMW structures by oxidative stress concomitantly leading to a functional switching from peroxidase to molecular chaperone [70]. Intriguingly, several other redox proteins in Arabidopsis, including thioredoxin reductase type C (NTRC) [71], GRXS17 [72], tetratricoredoxin (TDX) [73], and TRX-h3 [74], which play essential roles in oxidative and heat tolerance and exhibit a chaperone function, undergo conformational changes from LMW to HMW structures (Table 1). Further research is needed to determine how plants orchestrate APX and other redox-dependent chaperones (e.g., NTRC, 2-Cys PRX, TDX or TRXh3) to combat oxidative stress.

Table 1.

Structural and functional switching of Arabidopsis redox enzymes in response to redox state.

Protein	Protein structures and functions		Ref
	Reduction state	Oxidation state
APX1	LMW forms. Peroxidase activity.	HMW forms. Molecular chaperone.	[68]
2-Cys PRX	LMW forms. Peroxidase activity.	HMW forms. Chaperone.	[70]
NTRC	LMW forms. Disulfide reductase and foldase chaperone.	HMW forms. Holdase chaperone.	[71]
GRXS17	LMW forms. Involvement in the maturation of iron-sulfur proteins.	HMW forms. Holdase chaperone.	[72]
TDX	LMW forms. Disulfide reductase and foldase chaperone.	HMW forms. Holdase chaperone.	[73]
TRX-h3	LMW forms. Disulfide reductase activity.	HMW forms. Molecular chaperone.	[74]

2. Conclusions and outlook

Although much progress has been made in understanding the basic characteristics of APX isoenzymes, such as their distribution, structure and enzymological properties, there are still many unanswered questions. For example, the relative contribution of APXs in different cellular compartment is not entirely clear, and the functional crosstalk and overlap between APXs and other antioxidant enzymes also remain elusive. Besides its canonical H₂O₂ scavenging role, APX is likely to act as a H₂O₂ signaling regulator [75]. Chloroplastic APXs (tAPX and sAPX) are suggested to play a role in regulating retrograde H₂O₂ signal to modulate plant stress responses [76]. However, it is still unclear how much H₂O₂ is controlled by APXs. In order to further understand the H₂O₂ signaling, it is required to measure accurately H₂O₂ levels in different compartments using incisive redox sensors [77,78].

In addition to redox-based PTMs, nonoxidative PTMs also make contributions to altered APX activity in plants. Upon pathogen challenge, the wheat kinase start 1.1 is translocated to chloroplasts where it binds and phosphorylates tAPX, reducing its activity and ability to remove H₂O₂ [79]. Complete crotonylation at Lys136 of APX in chrysanthemum increases its enzymatic activity and further reduce the oxidative damage caused by low-temperature stress [80]. Notably, many of oxidative and nonoxidative PTMs are transient making comprehensive analysis under varied environmental stimuli difficult.

Despite their specificity towards ASC, APXs can also oxidize non-physiological aromatic substrates in vitro, such as p-cresol, o-dianisidine and guaiacol, at rates comparable to ASC [81]. Recent studies discussed in this review have demonstrated that APX activity also towards other substrates in plants, such as 4-coumarate, sinapyl alcohol, and GSH. It is not difficult to foresee that more potential substrates of APXs would be discovered in the future. The story of the multifaceted functions of APXs may have just begun!

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

No data was used for the research described in the article.