Cytosolic GAPDH: a key mediator in redox signal ...

DOI: 10.1007/s10535-017-0706-y BIOLOGIA PLANTARUM 61 (3): 417-426, 2017

417

REVIEW

Cytosolic GAPDH: a key mediator in redox signal transduction in plants S.S. YANG*and Q.H. ZHAI College of Life Sciences, Northwest A&F University, 712100, Yangling, Shaanxi, P.R. China Abstract Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serves not only as a key enzyme in glycolysis, but also as a multifunctional protein in other biological processes, especially in response to abiotic stresses in plants. Cytosolic GAPDH (GAPC) is a typical redox protein with selected catalytic cysteine, which undergoes reversible redox post-translational modifications (RPTMs) on its thiol group by reacting with hydrogen peroxide and nitric oxide related species. Moreover, the modified GAPC may interact with certain signal transmitters such as phosphatidic acid, phospholipase D, and osmotic stress-activated protein kinase. All these observations suggest that GAPC serve as a key mediator in redox signal transduction in plants. In this review, we provide an up-to-date insight into molecular mechanisms after H2O2- and NO-dependent oxidation of GAPC. We also discuss GAPC catalytic functions and potential functions as a modified protein by RPTMs.

Additional key words: abiotic stresses, cysteine modification, glycolysis, hydrogen peroxide, nitric oxide, post-translational modifications. Introduction Plants are constantly exposed to abiotic and/or biotic stresses, causing oxidative stress by accelerating the generation and accumulation of reactive oxygen species (ROS) and nitrogen species (RNS) (Huang et al. 2012, Schieber and Chandel 2014, Demidchik 2015, Noctor et al. 2015). Thus, to manage oxidation damage, higher plants have evolved fine-tuning mechanisms to sense and transfer redox signalling through reversible redox post-translational modifications (RPTMs) on some specific proteins (Shao et al. 2008, Rahantaniaina et al. 2013, Demidchik 2015, Sevilla et al. 2015). Some proteins with highly conserved cysteine residues are very sensitive to reactive species. Due to chemical properties of their thiol side chain, the catalytic

cysteine undergoes diverse reversible modifications (Winterbourn and Hampton 2008, Ferrer-Sueta et al. 2011, Akter et al. 2015). At physiological pH, the catalytic cysteine thiol group deprotonates into the thiolate anion (-S-) which is susceptible to be attacked by H2O2 and NO-related species, resulting in several types of oxidation forms, such as disulfides (SS). In addition, cysteine thiol group may undergo nitrosylsation (SNO), glutathionylation (SSG), and sulfenylation (SOH) (Spadaro et al. 2010, Go et al. 2015). In cells, these oxidized compounds can be reduced by reducing agents, such as glutathione (GSH), thioredoxin (Trx) and glutaredoxin (Grx) (Sevilla et al. 2015). Strikingly, the reactivity of cysteine thiol within protein toward these

Submitted 15 January 2016, last revision 16 September 2016, accepted 13 October 2016. Abbreviations: ABA - abscisic acid; ATG3 - autophagy-related protein; BaMV - bamboo mosaic virus; BPGA - 1,3-bisphospho-glyceric acid; DEA-NO - diethylamine NONOate; DTT - dithiothreitol; FER - FERONIA; GAPC - cytosolic GAPDH; GAPDH - glyceraldehyde-3-phosphate dehydrogenase; G3P - glyceraldehyde-3-phosphate; Grx - glutaredoxins; GSNO - nitrosoglutathione; MAP - mitogen-activated protein; NtOSAK -Nicotiana tabacum osmotic stress-activated protein kinase; OPP - oxidative pentose phosphate; OXI1 - oxidative signal-inducible1; PA - phosphatidic acid; PCD - programmed cell death; PLD - phospholipase D; QM - quantum mechanics; RNS - reactive nitrogen species; RPTM - redox post-translational modification; ROS - reactive oxygen species; SINAL7 - SEVEN IN ABSENTIA like7; SnRK2 - SNF1 (sucrose non-fermenting 1)-related protein kinase 2; SOH - sulphenylation; SSG - glutathionylation; Trx - thioredoxins. Acknowledgments: We are greatly acknowledged to the National Natural Science Foundation of China (Nos. 31271625 and 31671609), the State Key Laboratory of Soil Erosion and Dryland Farming on Loess Plateau, Institute of Soil and Water Conservation, Chinese Academy of Sciences (No. 10502), the National Basic Research Program of China (2015CB150402), and the National Natural Science Foundation of China (No. 51479189). * Corresponding author; e-mail: [email protected]

S.S. YANG, Q.H. ZHAI

418

reactive species is based on its acidity and nucleophilicity (Ferrer-Sueta et al. 2011) which are mainly determined by micro-environment of the protein reactive site, such as the pH value, three-dimensional structure of the protein, location of the selected cysteine, and its neighbouring residues (Roos et al. 2013, Go et al. 2015). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme of glycolysis where it reversibly converts glyceraldehyde-3-phosphate (G3P) coupled with its specific cofactor NAD+ and phosphate into NADH and 1,3-bisphosphoglycerate (1,3-BPG). It is a well-known multifunctional protein regulated by RPTMs on its selected catalytic cysteine residues in animal cells (Morigasaki et al. 2008, Tristan et al. 2011). The GAPDH is a tetramer of four identical or similar subunits. Whereas animal cells have only one homotetrameric form of GAPDH, plants possess three GAPDH subunits encoded by a gene family. A-subunit and B-subunit are encoded by gapA gene and gapB gene, respectively, and form A2B2- and A4-GAPDH isozymes, which serve as NADP-specific GAPDHs participating in photosynthetic CO2 fixation in chloroplasts (Brinkmann et al. 1989). The subunits of glycolytic homotetramer GAPDH isoforms (GAPC, GAPCp) are encoded by gapC and gapCp genes. These two isozymes are NAD-dependent GAPDHs and are located in the cytoplasm (GAPC) and in plastids (GAPCp), respectively (Petersen et al. 2003, Munoz-Bertomeu et al. 2009, 2010). All above mentioned types of GAPDH have a common feature of phosphorylating. The GAPC and GAPCp catalyze the phosphorylation of the substrate G3P in vitro, while A2B2- and A4-GAPDH catalyze the dephosphorylation of the substrate BPGA in vivo. In addition, plants contain a non-phosphorylating NADP-dependent GAPDH (np-GAPDH) in the cyto-plasm, catalyzing the oxidation of G3P directly to 3-phosphoglycerate (3PGA) (Rius et al. 2006). Like animal GAPDH, the plant cytosolic

glyceraldehyde-3-phosphate dehydrogenase (GAPC, EC 1.2.1.12,) was identified as a multi-functional protein (Wojtera-Kwiczor et al. 2013, Zachgo et al. 2013, Zaffagnini et al. 2013a). One important non-catalytic role of GAPC in plant cell is the response to various abiotic stresses, including salinity, drought, heat, cold, anaerobiosis, and cadmium toxicity (Jeong et al. 2000, Jiang et al. 2007, Baek et al. 2008, Wawer et al. 2010, Zhang et al. 2011, Guo et al. 2012, Krasensky and Jonak 2012). In addition, GAPC is also involved in control of the plant response to pathogen infection (Prasanth et al. 2011, Han et al. 2015, Henry et al. 2015). Under stress conditions, the transcription of GAPC is remarkably increased (Table 1). However, little is known about the detail molecular mechanism triggered by these stresses. Depending on the plant species, the gapc gene is further duplicated. For example Arabidopsis contains two gapc genes (GAPC1 and GAPC2). In recent years, several research groups have investigated the relationships of GAPC with H2O2 and NO and observed that GAPC is very sensitive towards H2O2. It also undergoes S-nitrosylation by nitroso-glutathione (GSNO, a cellular NO donor), leading to a strong inhibition of the enzyme activity and forming several redox-dependent modifications (Baek et al. 2008, Holtgrefe et al. 2008, Prasanth et al. 2011, Piattoni et al. 2013, Zaffagnini et al. 2013b, 2016, Piszczatowski et al. 2014), which may contribute to the function of GAPC in resistance to oxidative stress. A broader overview of GAPDH functioning as an information hub due to its property of a sensitive thiol switch has been published recently (Hildebrandt et al. 2015). In this review, we provide an up-to-date view on molecular mechanisms of GAPC undergoing H2O2- and NO-dependent oxidation, address GAPC catalytic functions and potential functions as a modified protein by RPTMs.

GAPC as a sensor of H2O2 Owing to its membrane diffusibility and relatively long life span, H2O2 functions as a signal molecule in H2O2-dependent signal transduction pathways via oxidizing critical thiols within redox-sensitive proteins. In the process of H2O2-dependent signal transduction, H2O2

selectively targets specific proteins. The oxidized proteins transfer the oxidation signal to their downstream signalling proteins through consecutive thiol-disulfide exchange reactions (Suzuki et al. 2012, Schmidt and Schippers 2015). Unfortunately, till today, questions of how H2O2 selectively recognizes its target proteins and what characteristics make these proteins become its target proteins, have not been fully answered. Several years ago, GAPC was found to be very sensitive protein to oxidative stress, which may underpin its role in plants under stress conditions (Schieber and Chandel 2014, Reczek and Chandel 2015, Schmidt and Schippers 2015, Niu and Liao 2016). Proteomics

approach to investigation of the H2O2 target proteins in cytosolic fraction in Arabidopsis thaliana showed that GAPC is the most prominent protein to H2O2 (Hancock et al. 2005). In vitro, H2O2 directly attacks the catalytic cysteine of GAPC causing a strong inhibition of the enzyme activity through three oxidation steps. Firstly, H2O2 converts the thiolate anion (-S-) of catalytic cysteine into sulfenate (-SO-) form that can be reduced back by dithiothreitol (DTT). Secondly, the sulfenate (-SO-) form reacts with another H2O2 giving rise to an irreversible oxidation of sulfinates (-SO2

-). Thirdly, the sulfinate (-SO2

-) experiences further oxidation into irreversible sulfonate (-SO3

-). Notably, the sulfenate (-SO-) form of GAPC can further react with reduced glutathione (GSH) into the S-glutathionylated form of GAPC. This form of GAPC can be also formed by the reaction of thiolate anion (-S-) with oxidized glutathione (GSSG) via thiol:disulfide exchange reaction. Moreover, the S-gluta-

REDOX SIGNAL TRANSDUCTION IN PLANTS

419

thionylated GAPC can be reduced by glutaredoxins (Grx) and thioredoxins (Trx) (Fig. 1) (Holtgrefe et al. 2008, Wawer et al. 2010, Guo et al. 2012, Piattoni et al. 2013). In addition, the substrate BPGA in the reverse reaction and the substrate G3P in the forward reaction can both protect the full enzyme activity from H2O2 inactivation by covalently acylating catalytic cysteine of GAPC (Bedhomme et al. 2012). These strong evidences suggest that a RPTMs mechanism might exist to regulate the

activity of GAPC in cellular environment. When a dramatic increase of H2O2 occurs in cells

under stress conditions, the fate of GAPC protein will either be irreversible oxidation, or conversion into glutathionylated form which is considered to be a protective molecular or -SO- form that is involved in other processes by interacting with and activating other cellular molecules.

Fig. 1. Molecular mechanisms of plant GAPC undergoing thiol-based redox modifications. Plant GAPC can be oxidized to sulfenicacid (GAPC-SOH) by one H2O2 molecule that can further react with GSH forming the glutathionylated form (GAPC-SSG). The sulfenic acid (GAPC-SOH) form can be further oxidized irreversibly to GAPC-SO2H. Both the GAPC-SOH and GAPC-SSG can be reduced by DTT, Trx, and Grx. In principle, GAPC-SO3H may also occur through an interaction of GAPC-SO2H with another H2O2

molecule, and GAPC-SSG may also be generated by GSSG with release of GSH, but the two reactions do not occur underphysiological conditions. GSSG can be formed by the reaction of GSH with ROS. Moreover, GAPC also undergoes S-nitrosylation by NO donors or trans-nitrosylation by GSNO generated by the reaction of GSH with NO-related species. Meanwhile, denitrosylation of nitrosylated GAPC is efficiently reversed by GSH with concomitant formation of GSNO. Investigations on the reactions of H2O2 with GAPDH isoforms in plants uncovered that GAPC is much more sensitive to H2O2 than other GAPDH isoforms. In wheat, GAPC is more sensitive to H2O2 than np-GAPDH, and its kinetic parameter of the oxidation is 63-fold higher than that for np-GAPDH (Piattoni et al. 2013). Although AtGAPC1 and AtGAPA contain an analogical acidic catalytic Cys149 in Arabidopsis, they show different responses to H2O2, e.g., the active Cys thiolate of AtGAPC1 reacts faster with H2O2 than AtGAPA, the conversion of sulfenic acid into sulfinic acid is also much faster in AtGAPC1 than in AtGAPA. Notably, sulfonic acid cannot be detected in this investigation. What is more, the oxidation of sulfinic to sulfonic acid would not occur in biological systems because of energy barrier

(Zaffagnini et al. 2016). There are some prerequisites for GAPC reactivity with H2O2. The GAPC is abundant enzyme in the cytoplasm. Its concentration is up to 240 μM, so the potential thiol content is estimated to 1 mM, which determines that GAPC has a higher content of active thiol than other redox proteins (Seidler et al. 2012). Furthermore, the analysis of GAPC crystal structure shows that the catalytic Cys occupies a large accessible surface area and is located in the N-terminal side of α-helix, which enhances the reaction rate and thiol acidity (Tien et al. 2012, Roos et al. 2013, Zaffagnini et al. 2016). Recently, in Arabidopsis, a ping-pong proton transfer mechanism has been described for favouring the acidity


420

and nucleophilicity of GAPC toward H2O2. In the model of GAPC active site by quantum mechanics (QM) formed by computation, the nitrogen of His176 has a role in neutralizing the negatively charged oxygen of catalytic Cys149. A set of neutral pair Cys149-(O)nH-His176 is formed as a stable intermediate to facilitate the multistep oxidation of the catalytic Cys149 by H2O2 (Zaffagnini et al. 2016). Additionally, it was found that a proton relay mechanism could explain the exceptional reactivity of human cytosolic GAPDH to H2O2. The proposed reaction mechanism is performed by thiolate and its neighbouring residues. Firstly, in the active-site of GAPDH, catalytic Cys152 thiolate and amide groups along with the γ-OH and amid groups of Thr153, hold H2O2 by H-bonds with the oxygen atoms of H2O2. Secondly, Cys152 thiolate takes SN2-type nucleophilic attacking on one of oxygen atom of H2O2, resulting in a hydroxyl anion generation that immediately takes a proton into water from Thr153

γ-OH, which accept a proton from Tyr 314 ζ-OH group. Finally, a proton from His179 is passed by the sulfenic acid on Cys152, the water molecule and the Thr153 hydroxyl group, reprotonated Tyr314. Thus, it is implied that GAPDH harbours a proton relay mechanism by promoting electron transfer to enhance the sensitivity of GAPDH to H2O2 (Peralta et al. 2015). The GAPC sequence contains two highly conserved cysteines in its catalytic domain. The counterpart of proton-relay residues, Thr153, Tyr314, Cys152, as well as His179 of human GAPDH are all also highly conserved in plants (Fig. 1 Suppl). Moreover, these residues in the structure of GAPC in rice show a similar position compared with that in AtGAPC1 and human GAPDH (Fig. 2) (Tien et al. 2012). Thus, we propose that plant cells have possibility to form a similar proton-relay like human cells.

Fig. 2. Representation of the catalytic sites of rice GAPC monomer O (PDB code 3E5R). The catalytic Cys 154 residue (C154), thecofactor NAD+ and its neighbouring residues are shown as sticks. The distances of the sulfur atom of catalytic Cys154 from the basicresidue His181 (H181), and the two sulfur atoms of Cys154 and Cys158 are indicated by dashed lines. Carbon - light blue, nitrogen -purple, oxygen - red, sulphur - yellow. GAPC and NO-related S-nitrosylation

S-nitrosylation is an important type of NO-dependent RPTMs. In cells, the S-nitrosylation occurs on specific cysteine residues and is triggered by RNS via oxidative mechanism or by S-nitrosothiol via trans-nitrosylation (Hess et al. 2005, Astier et al. 2011). Till present, several hundreds S-nitrosylated proteins have been identified in plants by proteomic approach (Lin et al. 2012, Wang

et al. 2015). Strikingly, S-nitrosylation appears to influence protein subcellular translocation and participate in plant immunity (Yu et al. 2012, Trapet et al. 2015). It is well confirmed that GAPC undergoes S-nitro-sylation. In vitro, the purified AtGAPC1 is subjected to S-nitrosylation by the strong chemical NO donor (diethylamine NONOate, DEA-NO) and trans-


421

nitrosylating agent (nitrosoglutathione, GSNO). Both reaction rate and extent of AtGAPC1 S-nitrosylation depend on the DEA-NO and GSNO concentrations. The S-nitrosylated AtGAPC1 can be reduced by DTT (Fig. 1) (Zaffagnini et al. 2013b). Interestingly, GSNO-dependent S-nitrosylation is slower than that DEA-NO-dependent, which is considered as a reason of higher steric hindrance of the cofactor NAD+ of AtGAPC1 for GSNO compared with NO. Meanwhile, the fact that the substrate 1,3-bisphosphoglycerate (1,3-BPG) can protect the enzyme from DEA-NO dependent S-nitrosylation suggests that Cys-149 may be the target of DEA-NO. Furthermore, through analysis of the nitrosylation of AtGAPC1 in the wild type and the site-specific mutant by biotin switch technique (BST), it is confirmed that AtGAPC1 is nitrosylated on catalytic Cys-149 (Zaffagnini et al. 2013b). In vivo, GAPC also undergoes S-nitrosylation in NO treated plants. In the nitric oxide excess1 (noe1) rice mutant, which has higher content of NO than the wild-type lines, GAPC is identified as a redox-related candidate for S-nitrosylation by biotin-switch assay and mass spectrometry method (Lin et al.

2012). Besides, in Arabidopsis cell cultures and leaves treated with GSNO and NO, respectively, GAPC is detected in S-nitrosylation state (Lindermayr et al. 2005). Further, like any RPTMs, S-nitrosylation is also completely reversed to ensure transient signalling. In cells, two mechanisms are responsible for recovering the S-nitrosylated proteins: reduced GSH and TRX system (Benhar et al. 2009, Sengupta and Holmgren 2013). The findings show that GAPC can be denitrosylated by GSH at physiological concentrations (2 mM), but not by plant cytoplasmic thioredoxins (Trx) (Fig. 1). Interestingly, the GSH-dependent denitrosylation activity depends on the GSH/GSNO ratio but is independent of the GSH/GSSG ratio (Zaffagnini et al. 2013b) MALDI-TOF mass spectrometry data show that GSNO also contributes to AtGAPC1 glutathionylation in a small fraction (Zaffagnini et al. 2013b). Thus, structural determinants allowing GSNO to act as a specific S-nitrosylation agent require further investigation. Moreover, the detail mechanism of GAPC S-nitrosylation in vivo remains to be understood as well.

Catalytic functions of GAPC The GAPC is the first enzyme in the second phase of glycolysis, that reversibly converts glyceraldehyde-3-phosphate coupled with its specific cofactor NAD+ and phosphate into 1,3-bisphosphoglycerate (1,3-BPG) and NADH. The 1,3-BPG owns a high-energetic phosphate group that might be transferred to ADP and generate ATP. This process is catalyzed by phosphoglycerate kinase (EC 2.7.2.3, PGKase) (Plaxton 1996). Thus, GAPC has some effect on other cellular metabolism requiring the source of ATP, NADH, and G3P. In cyanobacteria, the gap1 null strains produce NADPH via oxidative pentose phosphate (OPP) cycle, and gap1 over-expressing strains accumulate much more NADH (Kumaraswamy et al. 2013). In Arabidopsis, the gapc1 null mutant and the as-GAPC1 line exhibit slow growth and decreased amount of seeds due to abortions and empty embryonic sacs in siliques, which may be

caused by low ATP content and decreased respiration rate (Rius et al. 2008). Consistently, in another report, it has been found that AtGAPCs are directly associated with oil accumulation in seeds. The oil content as well as ATP and NAD(P)H content in the knockout lines of AtGAPCs were lower than in the wild type. In contrast, in AtGAPCs overexpressing lines, oil content in seeds is up to 42 % of dry mass and content of ATP and NAD(P)H is increased, which suggests that AtGAPCs may affect oil accumulation in seeds by controlling the content of ATP and NAD(P)H (Guo et al. 2014). Moreover, the expression of AtGAPC2 is significantly decreased under low-phosphate stress, and T-DNA insertion lines of gapC2 gene show hypersensitive phenotype to low-Pi stress (Wang et al. 2007), which may be caused by alteration of Pi content, because large amounts of Pi are associated with sugar metabolism and energy production.

Potential functions of GAPC as a modified protein Consistent with animal GAPDH, GAPC in plants also displays multiple functions beyond its metabolic roles. In A. thaliana, double knockout mutants of AtGAPCs show lower stomatal closure in response to ABA and higher H2O2 content under drought stress (Guo et al. 2012) than the wild type. Meanwhile, the mutant of phospholipase D (pldδ) also shows less stomatal closure in response to ABA, but without increase in H2O2 content (Guo et al. 2012). Under oxidative stress, GAPC turns into inactive form. Binding to PLD increases its activity (Guo et al. 2012). The plasma membrane-associated PLD hydrolyzes phospholipids to generate phosphatidic acid (PA) that

promotes stomatal closure in response to ABA. What is more, GAPC itself is a PA-binding protein, and the PLD deficiency lines have a higher rate of water loss by transpiration (Wang et al. 2006). Thus, it is a possible mechanism that GAPC senses the alteration of H2O2

content, transfers the redox signal to PLD, and the activated PLD promotes the stomatal closure by producing PA under drought (Guo et al. 2012). Besides, GAPC is translocated from the cytoplasm to nucleus under stress conditions in plants, which is a typical behaviour for moonlighting proteins. Under cold stress, GAPC was detected in the nucleus of Arabidopsis


422

(Bae et al. 2003). In cadmium-treated roots, inactivated GAPC has a steady accumulation in the nucleus along with increased NO content and cytosolic oxidation (Vescovi et al. 2013). Recently, the E3 ubiquitin-ligase SEVEN IN ABSENTIA like7 (SINAL7) was related with AtGAPC nuclear relocalization. In vitro, SINAL7 interacts with AtGAPC1 to mono-ubiquitinate AtGAPC1,

leading to lose of its catalytic activity, and AtGAPC1 is not found in the nucleus of the sinal7 mutant (Peralta et al. 2016). Although the mechanism of GAPC translocation and corresponding functions are not clear, its nuclear translocation implies that GAPC may have a moonlighting function.

Fig. 3. Functions of the modified GAPDH in redox signalling. GAPC is generally deprotonated into thiolate anion forms (GAPC-S-) in cells. Under abiotic stress, GAPC-S- is converted by H2O2 and NO into sulfenic acid (GAPC-SOH) and S-nitrosylation forms GAPC-SNO, respectively. The GAPC-SOH interacts with PLDδ to promote PA generation resulting in stomatal closure and MAPkinase cascades. The GAPC-SNO interacts with F-actin to regulate microtubule (MT) structure, while interaction with NtOSAK-Pi display translocation into nucleus. NtOSAK - Nicotiana tabacum osmotic stress activated kinase, NtOSAK-Pi - phosphorylated NtOSAK, PA - phosphatidic acid, PLD-δ - phospholipase D-δ; MT - microtubule. Moreover, in tobacco, NtGAPCs undergoes S-nitrosylation on its catalytic cysteine under salt stress, and co-immunoprecipitates with Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK) in extracts from salt-treated cells (Wawer et al. 2010). In plants, NtOSAK is a member of the sucrose non-fermenting 1-related protein kinase 2 (SnRK2) protein kinases family and is involved in NO signalling (Burza et al. 2006, Fujii et al. 2009, Fujita et al. 2009). Moreover, the interaction between NtGAPC and NtOSAK is observed both in the cytoplasm and nucleus. However, the mutated form of NtGAPCs lacking catalytic cysteine can also interact with NtOSAK but only in the cytoplasm. Therefore, catalytic cysteine S-nitrosylation is required for nuclear translocation of NtGAPCs but not for the interactions with NtOSAK (Wawer et al. 2010). In addition, GAPC has a great role in defence against phytopathogens. The GAPC prevents the replication of Bamboo Mosaic Virus (BaMV) in infected N. benthamiana (Prasanth et al. 2011). Besides, over-expression of GAPCs effectively restrains autophagy in N. benthamiana through interaction with autophagy-related protein 3 (ATG3) (Han et al. 2015). The ATG3 is a key component of autophagy and innate immunity in

plants, and its deficiency lines exhibit weak resistance to pathogen infection (Liu et al. 2005). Moreover, in gapc silence lines infected with Pseudomonas syringae pv. tabaci and P. syringae pv. tomato, the two pathogens were reduced to level compared with no infected plants (Han et al. 2015). Therefore, it is demonstrated that GAPCs might negatively regulate autophagy through directly suppressing the activity of ATG3. As it was previously shown, individual Arabidopsis GAPDH knockout lines exhibit enhanced disease resistance upon inoculation with the bacterial plant pathogen, and during the immune response, GAPC1 transcription increases by more than two-fold in comparison with the control, while other GAPDH isoform transcriptions are slightly down-regulated (Henry et al. 2015). Recently, a report has revealed that GAPC interactes with receptor protein kinase FERONIA (FER), which controls leaf starch accumulation in Arabidopsis. In FER deficiency lines, the activity of GAPC correspondingly decreases, but the amount of starch increases. Interestingly, the fer4 mutants mimic the gapc mutant (Yang et al. 2015). However, the mechanism of interaction between GAPC and FER needs further research.


423

Table 1 Genes of cytosolic GAPDH with adversity resistance

Gene Species Stress Reference

GAPC3 NP_001105385 Zea mays anaerobic and heat Russell et al. 1989 GAPC CAA55116 Craterostigma plantagineum dehydration and abscisic acid treatment Velasco et al. 1994 GAPC3 Q6K5G8 Oryza sativa salt Zhang et al. 2011 GAPC1 NP_187062 Arabidopsis thaliana salt , water, and cadmium Jiang et al. 2007

Guo et al. 2012 Vescovi et al. 2013

GAPC2 NP_172801 Arabidopsis thaliana

Water and low-phosphate Guo et al. 2012 Wang et al. 2007

GAPDH NP_001275344 Solanum tuberosum Phytophthora infestans salicylic acid

Laxalt et al. 1996

GAPC P09094 Nicotiana benthamiana salt Wawer et al. 2010 GAPC1 GAPC2 GAPC3

AKH15660.1 AKH15661.1 AKH15662.1

Nicotiana benthamiana Bamboo Mosaic Virus Tobacco Rattle Virus

Prasanth et al. 2011 Han et al. 2015

Conclusion and perspective

For a long period, GAPDH has been considered as a constitutive gene and has been used in gene expression analyses as a reference gene (Nicholls et al. 2012). However, accumulating evidence has shown that the transcription of GAPDH is dramatically induced by abiotic and biotic stresses (Zaffagnini et al. 2013a). In this manuscript, we summed up previous research to explain possible mechanisms of GAPC response to these stresses. The GAPC is equipped with the traits of a typical redox protein. The GAPC in plants has highly conserved cysteine residues, and its catalytic cysteine undergoes diverse reversible modifications, such as nitrosylation (SNO), glutathionylation (SSG), and sulfenylation (SOH). Meanwhile, these modifications can be reversed by GSH, Trx, and Grx (Holtgrefe et al. 2008, Wawer et al. 2010, Guo et al. 2012, Piattoni et al. 2013, Zaffagnini et al. 2013b). Moreover, GAPC harbours some specific prerequisites for its reactivity towards H2O2- and NO-related species. Notably, plant GAPC has a similar mechanism of reaction with H2O2 like animal GAPDH. Taken together, all of the evidence implies that GAPC is a novel candidate of a redox protein. Besides, the modified GAPC displays multi-function in some processes, including abiotic stress, leaf starch

accumulation, and pathogen infection, and has a nuclear relocalization. Thus, consistent with animal GAPDH, it is confirmed that GAPC is also a multifunctional protein regulated by RPTMs on its selected catalytic cysteine residue in plants. Under various abiotic stresses, plants sense environment changes immediately, and give rise to produce second messengers, such as ROS, RNS, and PA. Next, the signal molecules oxidize or bind some redox proteins and trigger MAP kinases cascade (Huang et al. 2012). For example, during water stress, H2O2 targets GAPC and promotes the interaction of GAPC and PLD to regulate stomatal closure (Guo et al. 2012). And under salt stress, the S-nitrosylated GAPC interacts with NtOSAK and activate MAP kinases cascade (Wawer et al. 2010). The ROS and RNS act as two key components in redox post-translational modifications (Reczek and Chandel 2015). The GAPC acts as a common target protein of ROS and RNS, which implies that GAPC may play a pivotal role in redox signalling (Fig. 3). In summary, the multi-function of GAPC is connected with its special molecular structure and redox properties. However, in comparison with well described animal GAPDH, the functional characterization of GAPC in plants is still at the beginning and further work is needed.

References Akter, S., Huang, J., Waszczak, C., Jacques, S., Gevaert, K.,

Van Breusegem, F., Messens, J.: Cysteines under ROS attack in plants: a proteomics view. - J. exp. Bot. 66: 2935-2944, 2015.

Astier, J., Rasul, S., Koen, E., Manzoor, H., Besson-Bard, A., Lamotte, O., Jeandroz, S., Durner, J., Lindermayr, C., Wendehenne, D.: S-nitrosylation: an emerging post-translational protein modification in plants. - Plant Sci. 181: 527-533, 2011.

Bae, M.S., Cho, E.J., Choi, E.Y., Park, O.K.: Analysis of the Arabidopsis nuclear proteome and its response to cold stress. - Plant J. 36: 652-663, 2003.

Baek, D., Jin, Y., Jeong, J.C., Lee, H.J., Moon, H., Lee, J., Shin, D., Kang, C.H., Kim, D.H., Nam, J., Lee, S.Y., Yun, D.J.: Suppression of reactive oxygen species by glyceraldehyde-3-phosphate dehydrogenase. - Phytochemistry 69: 333-338, 2008.

Bedhomme, M., Adamo, M., Marchand, C.H., Couturier, J.,


424

Rouhier, N., Lemaire, S.D., Zaffagnini, M., Trost, P.: Glutathionylation of cytosolic glyceraldehyde-3-phosphate dehydrogenase from the model plant Arabidopsis thaliana is reversed by both glutaredoxins and thioredoxins in vitro. - Biochem. J. 445: 337-347, 2012.

Benhar, M., Forrester, M.T., Stamler, J.S.: Protein denitrosylation: enzymatic mechanisms and cellular functions. - Nat. Rev. mol. cell. Biol. 10: 721-732, 2009.

Brinkmann, H., Cerff, R., Salomon, M., Soll, J.: Cloning and sequence analysis of cDNAs encoding the cytosolic precursors of subunits GapA and GapB of chloroplast glyceraldehyde-3-phosphate dehydrogenase from pea and spinach. - Plant mol. Biol. 13: 81-94,1989.

Burza, A.M., Pekala, I., Sikora, J., Siedlecki, P., Małagocki, P., Bucholc, M., Koper, L., Zielenkiewicz, P., Dadlez, M., Dobrowolska, G.: Nicotiana tabacum osmotic stress-activated kinase is regulated by phosphorylation on Ser-154 and Ser-158 in the kinase activation loop. - J. biol. Chem. 281: 34299-34311, 2006.

Demidchik, V.: Mechanisms of oxidative stress in plants: from classical chemistry to cell biology. - Environ. exp. Bot. 109: 212-228, 2015.

Ferrer-Sueta, G., Manta, B., Botti, H., Radi, R., Trujillo, M., Denicola, A.: Factors affecting protein thiol reactivity and specificity in peroxide reduction. - Chem. Res. Toxicol. 24: 434-450, 2011.

Fujii, H., Zhu, J.K.: Arabidopsis mutant deficient in 3 abscisic acid-activated protein kinases reveals critical roles in growth, reproduction, and stress. - Proc. nat. Acad. Sci. USA 106: 8380-8385, 2009.

Fujita, Y., Nakashima, K., Yoshida, T., Katagiri, T., Kidokoro, S., Kanamori, N., Umezawa, T., Fujita, M., Maruyama, K., Ishiyama, K., Kobayashi, M., Nakasone, S., Yamada, K., Ito, T., Shinozaki, K., Yamaguchi-Shinozaki, K.: Three SnRK2 protein kinases are the main positive regulators of abscisic acid signaling in response to water stress in Arabidopsis. - Plant Cell Physiol. 50: 2123-2132, 2009.

Go, Y.M., Chandler, J.D., Jones, D.P.: The cysteine proteome. -Free Radicals Biol. Med. 84: 227-245, 2015.

Guo, L., Devaiah, S.P., Narasimhan, R., Pan, X.Q., Zhang, Y.Y., Zhang, W.H., Wang, X.M.: Cytosolic glyceraldehyde-3-phosphate dehydrogenases interact with phospholipase Dδ to transduce hydrogen peroxide signals in the Arabidopsis response to stress. - Plant Cell 24: 2200-2212, 2012.

Guo, L., Ma, F., Wei, F., Fanella, B., Allen, D.K., Wang, X.M.: Cytosolic phosphorylating glyceraldehyde-3-phosphate dehydrogenases affect Arabidopsis cellular metabolism and promote seed oil accumulation. - Plant Cell 26: 3023-3035, 2014.

Han, S.J., Wang, Y., Zheng, X.Y., Jia, Q., Zhao, J.P., Bai, F., Hong, Y.G., Liu, Y.L.: Cytoplastic glyceraldehyde-3-phosphate dehydrogenases interact with ATG3 to negatively regulate autophagy and immunity in Nicotiana benthamiana. - Plant Cell 27: 1316-1331, 2015.

Hancock, J.T., Henson, D., Nyirenda, M., Desikan, R., Harrison, J., Lewis, M., Hughes, J., Neill, S.J.: Proteomic identification of glyceraldehyde 3-phosphate dehydrogenase as an inhibitory target of hydrogen peroxide in Arabidopsis. - Plant Physiol. Biochem. 43: 828-835, 2005.

Henry, E., Fung, N., Liu, J., Drakakaki, G., Coaker, G.: Beyond glycolysis: GAPDHs are multi-functional enzymes involved in regulation of ROS, autophagy, and plant immune responses. - PLoS Genet. 11: e1005199, 2015.

Hess, D.T., Matsumoto, A., Kim, S.O., Marshall, H.E., Stamler, J.S.: Protein S-nitrosylation: purview and parameters. - Nat.

Rev. mol. cell. Biol. 6: 150-166, 2005. Hildebrandt, T., Knuesting, J., Berndt, C., Morgan, B., Scheibe,

R.: Cytosolic thiol switches regulating basic cellular functions: GAPDH as an information hub? - Biol. Chem. 396: 523-537, 2015.

Holtgrefe, S., Gohlke, J., Starmann, J., Druce, S., Klocke, S., Altmann, B., Wojtera, J., Lindermayr, C., Scheibe, R.: Regulation of plant cytosolic glyceraldehyde 3-phosphate dehydrogenase isoforms by thiol modifications. - Physiol. Plant. 133: 211-228, 2008.

Huang, G.T., Ma, S.L., Bai, L.P., Zhang, L., Ma, H., Jia, P., Liu, J., Zhong, M., Guo, Z.F.: Signal transduction during cold, salt, and drought stresses in plants. - Mol. Biol. Rep. 39: 969-987, 2012.

Jeong, M.J., Park, S.C., Kwon, H.B., Byun, M.O.: Isolation and characterization of the gene encoding glyceraldehyde-3-phosphate dehydrogenase. - Biochem. biophys. Res. Commun. 278: 192-196, 2000.

Jiang, Y., Yang, B., Harris, N.S., Deyholos, M.K.: Comparative proteomic analysis of NaCl stress-responsive proteins in Arabidopsis roots. - J. exp. Bot. 58: 3591-3607, 2007.

Krasensky, J., Jonak, C.: Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks. - J. exp. Bot. 63: 1593-1608, 2012.

Kumaraswamy, G.K., Guerra, T., Qian, X., Zhang, S., Bryant, D.A., Dismukes, G.C.: Reprogramming the glycolytic pathway for increased hydrogen production in cyanobacteria: metabolic engineering of NAD+-dependent GAPDH. - Energy Environ. Sci. 6: 3722-3731, 2013.

Lin, A., Wang, Y., Tang, J., Xue, P., Li, C., Liu, L., Hu, B., Yang, F., Loake, G.J., Chu, C.: Nitric oxide and protein S-nitrosylation are integral to hydrogen peroxide-induced leaf cell death in rice. - Plant Physiol. 158: 451-464, 2012.

Lindermayr, C., Saalbach, G., Durner, J.: Proteomic identification of S-nitrosylated proteins in Arabidopsis. - Plant Physiol. 137: 921-930, 2005.

Liu, Y., Schiff, M., Czymmek, K., Tallóczy, Z., Levine, B., Dinesh-Kumar, S.P.: Autophagy regulates programmed cell death during the plant innate immune response. - Cell 121: 567-577, 2005.

Morigasaki, S., Shimada, K., Ikner, A., Yanagida, M., Shiozaki, K.: Glycolytic enzyme GAPDH promotes peroxide stress signaling through multistep phosphorelay to a MAPK cascade. - Mol. Cell 30: 108-113, 2008.

Munoz-Bertomeu, J., Cascales-Minana, B., Irles-Segura, A., Mateu, I., Nunes-Nesi, A., Fernie, A.R., Segura, J., Ros, R.: The plastidial glyceraldehyde-3-phosphate dehydrogenase is critical for viable pollen development in Arabidopsis. - Plant Physiol. 152:1830-1841, 2010.

Munoz-Bertomeu, J., Cascales-Minana, B., Mulet, J.M., Baroja-Fernandez, E., Pozueta-Romero, J., Kuhn, J.M., Segura, J., Ros, R.: Plastidial glyceraldehyde-3-phosphate dehydro-genase deficiency leads to altered root development and affects the sugar and amino acid balance in Arabidopsis. - Plant Physiol. 151: 541-558, 2009.

Nicholls, C., Li, H., Liu, J.P.: GAPDH: a common enzyme with uncommon functions. - Clin. exp. Pharmacol. Physiol. 39: 674-679, 2012.

Niu, L., Liao, W.: Hydrogen peroxide signaling in plant development and abiotic responses: crosstalk with nitric oxide and calcium. - Front. Plant Sci. 7: 230, 2016.

Noctor, G., Lelarge-Trouverie, C., Mhamdi, A.: The metabolomics of oxidative stress. - Phytochemistry 112: 33-53, 2015.

Peralta, D.A., Araya, A., Busi, M.V., Gomez-Casati, D.F.: The


425

E3 ubiquitin-ligase SEVEN IN ABSENTIA like 7 mono-ubiquitinates glyceraldehyde-3-phosphate dehydrogenase 1 isoform in vitro and is required for its nuclear localization in Arabidopsis thaliana. - Int. J. Biochem. cell. Biol. 70: 48-56, 2016.

Peralta, D., Bronowska, A.K., Morgan, B., Dóka, É., Van, L.K., Nagy, P., Gräter, F., Dick, T.P.: A proton relay enhances H2O2 sensitivity of GAPDH to facilitate metabolic adaptation. - Nat. chem. Biol. 11:156-163, 2015.

Petersen, J., Brinkmann, H., Cerff, R.: Origin, evolution, and metabolic role of a novel glycolytic GAPDH enzyme recruited by land plant plastids. - J. mol. Evol. 57: 16-26, 2003.

Piattoni, C.V., Guerrero, S.A., Iglesias, A.A..: A differential redox regulation of the pathways metabolizing glyceraldehyde-3-phosphate tunes the production of reducing power in the cytosol of plant cells. - Int. J. mol. Sci. 14: 8073-8092, 2013.

Piszczatowski, R.T., Rafferty, B.J., Rozado, A., Tobak, S., Lents, N.H.: The glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) is regulated by myeloid zinc finger 1 (MZF-1) and is induced by calcitriol. -Biochem. biophys. Res. Commun. 451: 137-141, 2014.

Plaxton, W.C.: The organization and regulation of plant glycolysis. - Annu. Rev. Plant Biol. 47: 185-214, 1996.

Prasanth, K.R., Huang, Y.W., Liou, M.R., Wang, R.Y., Hu, C.C., Tsai, C.H., Meng, M., Lin, N.S., Hsu, Y.H.: Glyceraldehyde 3-phosphate dehydrogenase negatively regulates the replication of Bamboo mosaic virus and its associated satellite RNA. - J. Virol. 85: 8829-8840, 2011.

Rahantaniaina, M.S., Tuzet, A., Mhamdi, A., Noctor, G.: Missing links in understanding redox signaling via thiol/disulfide modulation: how is glutathione oxidized in plants? - Front. Plant Sci.4: 477, 2013.

Reczek, C.R., Chandel, N.S.: ROS-dependent signal transduction. - Curr. Opin. Cell Biol. 33: 8-13, 2015.

Rius, S.P., Casati, P., Iglesias, A.A., Gomez-Casati, D.F.: Characterization of an Arabidopsis thaliana mutant lacking a cytosolic non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase. - Plant mol. Biol. 61: 945-957, 2006.

Rius, S.P., Casati, P., Iglesias, A.A., Gomez-Casati, D.F.: Characterization of Arabidopsis lines deficient in GAPC-1, a cytosolic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase. - Plant Physiol.148: 1655-1667, 2008.

Roos, G., Foloppe, N., Messens, J.:Understanding the pK(a) of redox cysteines: the key role of hydrogen bonding. - Antioxid. Redox Signal. 18: 94-127, 2013.

Schieber, M., Chandel, N.S.: ROS function in redox signaling and oxidative stress. - Curr. Biol. 24: R453-R462, 2014.

Schmidt, R., Schippers, J.H.: ROS-mediated redox signaling during cell differentiation in plants. - Biochim. biophys Acta 1850: 1497-1508, 2015.

Seidler N.W.: GAPDH: Biological Properties and Diversity. - Springer, Dordrecht 2012.

Sengupta, R., Holmgren, A.: Thioredoxin and thioredoxin reductase in relation to reversible S-nitrosylation. - Antioxid. Redox Signal. 18: 259-269, 2013.

Sevilla, F., Camejo, D., Ortiz-Espin, A., Calderon, A., Lazaro, J.J., Jimenez, A.: The thioredoxin/peroxiredoxin/ sulfiredoxin system: current overview on its redox function in plants and regulation by reactive oxygen and nitrogen species. - J. exp. Bot. 66: 2945-2955, 2015.

Shao, H.B., Chu, L.Y., Shao, M.A., Jaleel, C.A., Mi, H.M.: Higher plant antioxidants and redox signaling under

environmental stresses. - Compt. rend. Biol. 331: 433-441, 2008.

Spadaro, D., Yun, B.W., Spoel, S.H., Chu, C., Wang, Y.Q., Loake, G.J.: The redox switch: dynamic regulation of protein function by cysteine modifications. - Physiol. Plant. 138: 360-371, 2010.

Suzuki, N., Koussevitzky, S., Mittler, R., Miller, G..: ROS and redox signalling in the response of plants to abiotic stress. - Plant Cell Environ. 35: 259-270, 2012.

Tien, Y.C., Chuankhayan, P., Huang, Y.C., Chen, C.D., Alikhajeh, J., Chang, S.L., Chen, C.J..: Crystal structures of rice (Oryza sativa) glyceraldehyde-3-phosphate dehydro-genase complexes with NAD and sulfate suggest involvement of Phe37 in NAD binding for catalysis. - Plant mol. Biol. 80: 389-403, 2012.

Trapet, P., Kulik, A., Lamotte, O., Jeandroz, S., Bourque, S., Nicolas-Frances, V., Rosnoblet, C., Besson-Bard, A., Wendehenne, D.: NO signaling in plant immunity: a tale of messengers. - Phytochemistry 112: 72-79, 2015.

Tristan, C., Shahani, N., Sedlak, T.W., Sawa, A.: The diverse functions of GAPDH: views from different subcellular compartments. - Cell. Signal. 23: 317-323, 2011.

Vescovi, M., Zaffagnini, M., Festa, M., Trost, P., Lo Schiavo, F., Costa, A.: Nuclear accumulation of cytosolic glyceraldehyde-3-phosphate dehydrogenase in cadmium-stressed Arabidopsis roots. - Plant Physiol.162: 333-346, 2013.

Wang, P., Du, Y., Hou, Y.J., Zhao, Y., Hsu, C.C., Yuan, F., Zhu, X., Tao, W.A., Song, C.P., Zhu, J.K.: Nitric oxide negatively regulates abscisic acid signaling in guard cells by S-nitrosylation of OST1. - Proc. nat. Acad. Sci. USA 112: 613-618, 2015.

Wang, X.Y., Chen, Y.F., Zou, J.J., Wu, W.H.: Involvement of a cytoplasmic glyceraldehyde-3-phosphate dehydrogenase GapC-2 in low-phosphate-induced anthocyanin accumulation in Arabidopsis. - Chin. Sci. Bull. 52: 1764-1770, 2007.

Wang, X., Devaiah, S.P., Zhang, W., Welti, R.: Signaling functions of phosphatidic acid. - Progr. Lipid Res. 45: 250-278, 2006.

Wawer, I., Bucholc, M., Astier, J., Anielska-Mazur, A., Dahan, J., Kulik, A., Wyslouch-Cieszynska, A., Zareba-Koziol, M., Krzywinska, E., Dadlez, M., Dobrowolska, G., Wendehenne, D.: Regulation of Nicotiana tabacum osmotic stress-activated protein kinase and its cellular partner GAPDH by nitric oxide in response to salinity. - Biochem. J. 429: 73-83, 2010.

Winterbourn, C.C., Hampton, M.B.: Thiol chemistry and specificity in redox signaling. - Free Radicals Biol. Med. 45: 549-561, 2008.

Wojtera-Kwiczor, J., Gross, F., Leffers, H.M., Kang, M., Schneider, M., Scheibe, R.: Transfer of a redox-signal through the cytosol by redox-dependent microcompart-mentation of glycolytic enzymes at mitochondria and actin cytoskeleton. - Front. Plant Sci. 3: 1-19, 2013.

Yang, T., Wang, L., Li, C., Liu, Y., Zhu, S., Qi, Y., Liu, X., Lin, Q., Luan, S., Yu, F.: Receptor protein kinase FERONIA controls leaf starch accumulation by interacting with glyceraldehyde-3-phosphate dehydrogenase. - Biochem. biophys. Res. Commun. 465: 77-82, 2015.

Yu, M.,Yun, B.W., Spoel, S.H., Loake, G.J.: A sleigh ride through the SNO: regulation of plant immune function by protein S-nitrosylation. - Curr. Opin. Plant Biol. 15: 424-430, 2012.

Zachgo, S., Hanke, G.T., Scheibe, R.: Plant cell micro-


426

compartments: a redox-signaling perspective. - Biol. Chem. 394: 203-216, 2013.

Zaffagnini, M., Fermani, S., Calvaresi, M., Orru, R., Iommarini, L., Sparla, F., Falini, G., Bottoni, A., Trost, P.: Tuning cysteine reactivity and sulfenic acid stability by protein microenvironment in glyceraldehyde-3-phosphate dehydro-genases of Arabidopsis thaliana. - Antioxid. Redox Signal. 24: 502-517, 2016.

Zaffagnini, M., Fermani, S., Costa, A., Lemaire, S.D., Trost, P.: Plant cytoplasmic GAPDH: redox post-translational modifi-cations and moonlighting properties. - Front. Plant Sci. 4:

450, 2013a. Zaffagnini, M., Morisse, S., Bedhomme, M., Marchand, C.H.,

Festa, M., Rouhier, N., Lemaire, D., Trost, P.: Mechanisms of nitrosylation and denitrosylation of cytoplasmic glyceraldehyde-3-phosphate dehydrogenase from Arabidopsis thaliana. - J. biol. Chem. 288: 22777-22789, 2013b.

Zhang, X.H., Rao, X.L., Shi, H.T., Li, R.J., Lu, Y.T.: Over-expression of a cytosolic glyceraldehyde-3-phosphate dehydrogenase gene OsGAPC3 confers salt tolerance in rice. - Plant Cell Tissue Organ Cult. 107: 1-11, 2011.

Cytosolic GAPDH: a key mediator in redox signal ...

Documents