Redox biology response in germinating Phaseolus vulgaris seeds exposed to copper: Evidence for differential redox buffering in seedlings and cotyledon

In agriculture, heavy metal contamination of soil interferes with processes associated with plant growth, development and productivity. Here, we describe oxidative and redox changes, and deleterious injury within cotyledons and seedlings caused by exposure of germinating (Phaseolus vulgaris L. var. soisson nain hâtif) seeds to copper (Cu). Cu induced a marked delay in seedling growth, and was associated with biochemical disturbances in terms of intracellular oxidative status, redox regulation and energy metabolism. In response to these alterations, modulation of activities of antioxidant proteins (thioredoxin and glutathione reductase, peroxiredoxin) occurred, thus preventing oxidative damage. In addition, oxidative modification of proteins was detected in both cotyledons and seedlings by one- and two-dimensional electrophoresis. These modified proteins may play roles in redox buffering. The changes in activities of redox proteins underline their fundamental roles in controlling redox homeostasis. However, observed differential redox responses in cotyledon and seedling tissues showed a major capacity of the seedlings’ redox systems to protect the reduced status of protein thiols, thus suggesting quantitatively greater antioxidant protection of proteins in seedlings compared to cotyledon. To our knowledge, this is the first comprehensive redox biology investigation of the effect of Cu on seed germination.

In plants, modulation of cellular redox homeostasis involves three principal systems: glutaredoxin (Grx)/GSH/GR, thioredoxin/NADPH/NADPH-dependent thioredoxin reductase (EC 1.6.4.5) (Trx/NADPH/ NADPH-dependent thioredoxin reductase (NTR); EC 1.8.1.9) [16][17][18], and ferredoxin-NADP oxido-reductase (FNR, EC 1.18.1.2). Functioning of these redox system components is based on their redox activity [19,20]. In germinating seeds, their roles have previously been investigated [21,22]. Indeed, Trx has been shown to be involved in extensive changes in the redox state of major storage proteins, the thiols of which are converted from the oxidized (-S-S-) form to reduced (-SH) forms [21]. They also play a role in activation of proteases and inactivation of protease inhibitors [20][21][22]. Under normal conditions, the intracellular redox state is predominantly reducing, but processes such as oxidative stress, notably under abiotic-and biotic-induced stress, can shift the redox balance toward an oxidizing state [23][24][25]. Regulation of the redox status of proteins, including the sulfhydryl groups of cysteine, can act as a "switch" for the activity of proteins involved in specific signaling events and in cell cycle control [26].
Some of the recognized effects of Cu toxicity on bean seeds are: (1) alteration of germinative metabolism [27,28]; and (2) disruption of the capacity of the ubiquitin-proteasome pathway to eliminate oxidatively-damaged proteins [29]. Besides, in cells, Cu exists in either a reduced (Cu + ) or an oxidized (Cu 2+ ) state, which makes Cu a redox-active metal by the induction of electron-transfer reactions. This redox-activity can also promote the generation of reactive oxygen radicals and affect every category of macromolecule. On these grounds, the present work aimed to shed more light on the mechanism of Cu-induced toxicity and on the cell defense response in bean cotyledons and seedlings. In particular, we are interested in elucidating changes in antioxidative enzymes (SOD, CAT, APX, POX and GPX) and enzymes of NAD (P)H-recycling dehydrogenases (G6PDH, 6PGDH and MDH) under Cu-induced stress. In addition, effects of Cu on the coenzyme pattern, NAD(P)H oxidase (EC 1.6.99.6; EC 1.6.99.3) activity and redox components (Grx, GR, Trx, NTR, Fd, FNR and peroxiredoxin (Prx; EC 1.11.1.15) were investigated.

Germination and copper treatment conditions
Seeds of the bean (Phaseolus vulgaris L. var. soisson nain hâtif) were germinated at 25 ± 1.5˚C in the darkness in the presence of H 2 O or 200 μM CuCl 2 , according to Karmous et al. [27]. Whole seedlings and cotyledons were collected, respectively, at days 3 and 9.
Antioxidative and redox response in bean seeds exposed to Cu

Measurement of oxidative indicators
H 2 O 2 levels were measured according to Sergiev et al. [30]. Carbonyl and thiol groups were determined according to the methods of Reznick and Packer [31] and Ellman [32], respectively. Measurements were performed using 0.5 mL of cotyledon or seedling extract in a total reaction volume of 2 mL.

Coenzyme extraction and measurement
Reduced (NADPH and NADH) and oxidized (NADP + and NAD + ) forms of coenzyme were extracted according to the method of Zhao et al. [44], respectively, in 0.1 M NaOH and 0.1 M HCl, followed by centrifugation at 20,000 × g at 10 min at 4˚C. Twenty μL of cotyledon or seedling extract in 500 μL total reaction volume were used for quantification according to the procedures described by Matsumura and Miyachi [45].

Statistical analysis
All experiments were performed at least in triplicate. Values are means ± standard error SE, of three technical and five biological replicates. These were compared for significance of differences at p < 0.05 using the ANOVA test followed by Student's t test analysis. Images of 2D gels were subjected to landmarking alignment so that corresponding spots were matched with each other. This models protein spots mathematically as a 3D Gaussian distribution and determines maximum absorption after raw image correction and background subtraction. Spot intensities were normalized to make the total density in each gel image equal, and quantitative and qualitative analyses were performed. The protein spots were detected automatically and then edited manually to remove streaks, speckles, and artifacts. Two D gels were replicated at least three times and the results reported as means ± SD. Analyses of variance (one-way ANOVA) followed by Tukey's post hoc multiple comparison tests were performed using the software package Statistica 8.0 to compare Cu-treated tissues with controls. Statistically significant differences between all spots in 2D gel image were established at p<0.05 and assessed using Student's t test.

Effects of copper on seed germination
Cu strongly inhibited germination of bean seeds, as evidenced by decreased growth of the Cutreated seedlings over 9 days (Fig 1). A two-day delay in germination was evident in Cu-treated seeds (Fig 2A and 2B). However, the seedling length showed drastic decrease with increasing Cu concentration (Fig 2C-2F). Nevertheless, for the present study, we chose 200 μM Cu as our working concentration and days 3 and 9 for, respectively, seedlings and cotyledons.
Response of antioxidant enzyme systems to copper-induced stress A significant increase of H 2 O 2 content was observed in Cu-treated cotyledon extracts although no significant increase was evident in seedling extracts after 3 days' treatment (Table 1). In S1 Appendix, we also recorded an increase in MDA levels in both tissues after exposure to Cu. Hence, we were interested to ascertain the mechanisms by which bean seeds respond to Cuinduced stress. Indeed, marked enhancement of the antioxidant enzymatic activities; SOD, CAT and peroxidases (APX, GPX and POX) in seedlings (Table 1) and cotyledons (Table 1) were evident after Cu treatment. This increase was significant for all antioxidant enzymes (except SOD and APX in cotyledons), as compared to controls. In addition, time courses of enzyme activities suggested that, in seedlings, SOD and CAT activities increased after only 4 hours of germination while POX, APX and GPX increased after 24 hours (Fig 3). In cotyledons, SOD, CAT and APX activities increased from the first day of germination, with more significant activation at days 3, 6 and 9 (Fig 3). However, GPX and POX showed increased activities after day 3.
Upon Cu treatment, activities of NAD(P)H-independent dehydrogenases, notably, G6PDH, 6PGDH and MDH were significantly enhanced in both cotyledons and seedlings (Table 1). These biochemical observations led us to examine changes in protein redox status in response to Cu exposure, as well as possible relationships between protein thiol management and thiol-dependent enzymatic redox systems.

Redox changes under copper-induced stress
Levels of both CO and -SH groups were higher in Cu-treated seedlings whilst, in cotyledons, an increase in CO level versus a net decline in level of protein -SH was observed (Table 2). This suggested that protein thiol status was affected by oxidation due to Cu in both organs. In addition, when compared to respective controls, cotyledons of Cu-treated seeds showed a significant decrease in Trx activity, but no significant variation in Grx activity and a marked increase in GR and NTR activities (Table 3). However, in seedlings, a significant increase in the activities of NTR and Trx was evident with no significant increase in GR and Grx activities in the presence of Cu (Table 3). These data suggest that, as a consequence of Cu-induced stress, there is a more protective effect of Grx/GR and Trx/NTR systems on protein thiols in seedlings than in cotyledons. Curiously, the Fd/FNR system was highly stimulated in seedlings whilst it was inhibited in cotyledons (Table 3). Prx activity also increased in both seedlings and cotyledons, as compared with controls, which may implicate this enzyme in Cu defense.
To assess potential contributions of coenzyme forms in response to Cu-induced stress, possible changes in total quantities of nicotinamide coenzymes (oxidized form + reduced form) were examined. The enzymatic activities responsible for oxidation of the reduced forms of coenzyme were also measured. A net increase in total coenzyme levels was found in both cotyledons and seedlings ( Table 4). The redox ratio of coenzymes (NADP + /NADPH and NAD + /NADH), as well as NADPH oxidase activity increased significantly in seedlings, whilst only the NAD + /NADH ratio and NADH oxidase activity increased significantly in cotyledons. These findings suggested elevated levels of oxidized coenzyme forms (NADP + and NAD + ) in response to Cu treatment, as compared with controls (Table 4). total proteins showed 1,174 and 599 spots, respectively, in seedlings and cotyledons (Fig 6; Table 5). Amongst these, 77 and 34, respectively, were significantly modified (±1.5-fold), compared to controls (p<0.05). It was noted in these preparations that the majority of proteins focused in the pI range 5-10 and in the M r range 15-75 kDa. Comparison of spot patterns between Cu-treated and control samples revealed more increase than decrease of proteins, in the presence of Cu in both tissues, suggesting activation of biosynthesis upon heavy metal exposure. Fluorescence intensity measurements after IAF labeling showed 4 and 27 spots of interest, respectively, containing -SH groups (p< 0.05 and > = 1.5-fold). In cotyledons, all the proteins corresponding to 4 spots seemed to be increased in abundance whilst, in the seedlings, no significant variation was detected between replicates in the presence of Cu (13 increases vs 14 decreases, Fig 6). Figs 7 and 8 showed an increase in the total CO, respectively, in the seedlings and the cotyledons after Cu exposure. Carbonylation is known to be a general indicator of protein oxidation [6,32]. These findings were corroborated by 2D gel analysis using FTSC-specific fluorescence. The representative 2D gels of CO groups of proteins showed 610 and 356 total protein spots, respectively, in cotyledons and seedlings. Among these, 234 and 159 corresponded with spots detected by fluorescence after FTSC labeling ( Table 6). The interesting spots (significant at p<0.05 and > = 1.5 fold), 29 and 3 spots respectively, in cotyledons and seedlings (Fig 9) also showed more increase than decrease by Cu, suggesting enhanced protein biosynthesis under Cu-induced stress.

Discussion
Because of their sessile character, plants confront various environmental stresses during their life cycles. Heavy metals, particularly Cu, represent a serious problem in agricultural production [48]. Research into germination and seedling growth (post-germination phase) are considered fundamental to evaluating the toxic effects of heavy metals on important agronomic plants such as beans [49]. It has been suggested that vulnerability of bean seeds towards copper stress can be partly explained by disruption of metabolic pathways affecting seedling growth [27,28]. In the present work, a significant delay in seedling growth (Figs 1 and 2) was shown to be associated with metabolic disturbances possibly occurring in both seedlings and cotyledons. In fact, investigation of the changes in antioxidant metabolism and cellular redox status confirmed that Cu induced intrinsic production of ROS, notably H 2 O 2 ( Table 1). Many heavymetal-stressed species have been reported to defend against ROS overproduction [50][51][52]. In the present work, the formation of H 2 O 2 seems to be mediated by the redox-active Cu. Therefore, metal ions-catalyzed reactive oxygen radicals might be potent mediators of the cellular oxidative injury, which can damage proteins, nucleic acids, and lipids. Indeed, in addition to lipid peroxidation (see increased malondialdehyde levels in S1 Appendix), we aimed to investigate mainly changes affecting proteins. Cu exhibits an affinity for the sulfur in cysteine or methionine, and Cu 2+ binds to oxygen or imidazole nitrogen groups of aspartic and glutamic acid, or histidine. In addition, Cu can displace other metals, such as zinc, from their cognate Table 2 ligands in metalloproteins, which can result in inappropriate protein structures or inhibition of activity of many important cellular enzymes. Additionally, generation of oxidative stress has been reported in germinating legume seeds after heavy metal exposure [53,54]. Here, endogenous H 2 O 2 accumulation, triggers stimulation of antioxidant enzymes SOD, CAT and peroxidases (APX, GPX and POX), thus allowing enhanced elimination of H 2 O 2 in seedlings and cotyledon tissues after Cu exposure (Table 1; Fig 3). Enzymatic antioxidative response differs between seedlings and cotyledons, however, with respect to the order of activation of the antioxidative enzymes during germination (Figs 3  and 4). Changes in antioxidant concentrations and activities of ROS-processing enzymes have been associated with seed germination under heavy metals [55]. POXs are considered to be heavy-metal stress-related enzymes and are sometimes used as stress markers in metal poisoning scenarios [13]. Effects of heavy metals on antioxidant enzyme activities and their involvement in defense mechanisms against oxidative damage have been widely reported in the literature, but remain controversial and vary amongst plant species, different tissues and varying exposure regimes [3,13,53,56]. Cu also inhibits some enzymes such as acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2), G6PDH, isocitrate dehydrogenase, CAT, GPX and glutathione transferases. Antioxidant systems are likely to be involved in defense against heavy metal-imposed oxidative stress, but might also be direct biochemical targets for metallic ion-induced toxicity.

. Levels of protein carbonyl and thiol groups in the seedlings (3 days-old) and the cotyledons (9 days-old) of germinated bean seeds in the presence of H 2 O (CTR) or 200 μM
In addition to their enzymatic antioxidant capacity, plant tolerance to heavy metal-induced toxicity depends crucially on the availability of reduced cofactors, such as NAD(P)H [13,14].
The key antioxidant and redox systems such as Trx, Grx and the Asc-GSH cycle depend heavily on NADPH rather than NADH for reducing equivalents. Hence, in response to oxidative stress, cells may need to shift from pathways producing NADH to others producing NADPH, such as the pentose phosphate pathway [15]. In this regard, increased activity of NAD(P)H-independent dehydrogenases, notably G6PDH, 6PGDH and MDH, in both seedlings and cotyledons (Table 1) after Cu treatment, most likely enable increased availability of NADPH to stressed cells [14,54,55]. Values are means ± SE (n = 5). Total coenzymes (oxidized and reduced forms) were expressed in nmol/g fresh weight, and activities of NAD(P)H oxidases were expressed in mU/mg proteins. Asterisks indicate significant differences compared with the respective control sample * p < 0.05, Antioxidative and redox response in bean seeds exposed to Cu In the current study, protein redox status and the major intracellular redox actors that control formation/reduction of intra-and/or inter-molecular disulfide bridges were also studied. Analysis of the components of different redox systems suggests that, in cotyledons, neither the Trx/NTR nor Grx/GR systems were involved in improving the protection of protein thiols to oxidation, possibly due to direct inhibition by Cu ions of the redox enzymes. Cu also seems to induce differential redox responses in cotyledons and seedlings. In fact, it seems that both Trx and Grx enzymes had not improved the redox status of thiols in cotyledons. However, in seedlings the levels of all components of the redox systems were elevated, thus suggesting a contribution of Trx/NTR/NADPH, despite the vulnerability of the coenzymes to enzymatic oxidation. A decreased level of reduced protein thiols was found coupled with increased  (Table 2), indicating extensive protein oxidation [29]. But in seedlings, despite an increase in protein carbonyl content, enhanced protein thiol levels ( Table 2) suggest that thiol status is protected via Trx and Grx activities (Table 3). Redox systems are thought to play fundamental roles in controlling plant redox and defense status when subjected to abiotic stress [18,24,25]. Indeed, Trx are involved in protection against oxidative damage by regeneration of Prxs and methionine sulphoxide reductases, allowing detoxification of various peroxides and protein repair [57]. Trx h can act as a hydrogen donor for the peroxiredoxin-1 Cys, which protects macromolecules in seedlings against oxidation during the early phases of imbibition [58]. In cotyledons, loss of Fd and FNR activities may generate intracellular oxidative stress whilst, in seedlings, the Fd/FNR system appears to be involved in modulation of redox status (Table 3). On the other hand, increased Prx activity in both seedlings and cotyledons after Cu exposure suggest an antioxidant role, most probably via POX activity using H 2 O 2 , peroxynitrite and hydroperoxides as substrates [59].

Table 4. Activities of NAD(P)H oxidases and redox ratios of coenzymes in the seedlings (3 days-old) and the cotyledons (9 days-old) of germinated bean seeds in the presence of H 2 O (CTR) or 200 μM
In response to Cu stress, high levels of oxidized coenzymes compared to reduced ones accumulated in seedling and cotyledon tissues (Table 4), despite increased NAD(P)H-independent dehydrogenase activities. This observation is most likely due to enhanced consumption of NADPH following the induction of NTR activity in cotyledons and both NTR and GR activity in seedlings. Another explanation could be stimulation of enzymes oxidizing reduced coenzymes. For example, enhanced NAD(P)H oxidase activity could result in elevation of oxidized forms of coenzymes at the expense of reduced ones, thus increasing NAD(P) + /NAD(P)H ratios [53].
Cu-induced biochemical disturbances in germinating bean seeds, including modulation of activities of antioxidant enzymes, could prevent oxidative damage. However, differential redox responses in cotyledon and seedling tissues suggest a major capacity of redox systems to prevent oxidation of protein thiols in seedlings in particular. Protein thiol status of seedlings was not affected by Cu with an apparent increase in the reduced SH pool (Tables 3 and 4). This could be explained by higher Trx and Grx activities on protein thiols, underlining these proteins' fundamental rules in controlling seedling redox homeostasis (despite other antioxidative alterations also described here). Recycling of protein thiols also appears to occur mainly via redox systems (Trx/NTR), (Grx/GR), (Fd/FNR) and Prx, rather than via NADPH. These results are corroborated by the study of proteomic changes occurring to SH and CO groups of proteins in both cotyledon and seedling.  Simultaneous profiling of many proteins represents a novel way to compare dynamic responses towards heavy metals [60]. Proteomics is increasingly used to detect effects of environmental contaminants in ecotoxicology [61,62]. 2DE analysis revealed significant changes in abundance of SH and CO groups of protein species in cotyledons and the seedlings of Custressed bean seeds (Figs 6-9). Oxidative stress can trigger conformational changes making protein thiols more reactive towards cationic groups and modifying their susceptibility to oxidation, either by increasing their exposure to the matrix or by decreasing side-chain pKa values [63]. In addition, transitory oxidative stress may increase protection of thiols, e.g. by glutathionylation or formation of sulphenic acid [64]. Heavy metals disrupt the majority of cellular mechanisms as well as causing differential accumulation of proteins involved in the regulation of cellular redox status [65]. Decreased availability of the main proteins involved in important cellular processes has also been reported [66].

Total
In the present study, we have profiled the role of a network of ROS-detoxifying enzymes in protecting bean seeds from Cu-induced stress. Whilst antioxidant protection mechanisms have an important role in Cu stress tolerance in both cotyledons and seedlings, we have discovered subtle differences in the two organs. Notably, we found a greater capacity for protein protection in seedlings compared to cotyledons.
One of the likely ways for ROS to interact with proteins is through thiol modification of cysteine residues, which can be oxidised to varying degrees triggering changes in protein conformation and activity [67]. Redox signalling in higher plants may include the activation of the mitogen activated protein kinase cascade, inhibition of phosphatases and activation of Ca 2+ channels and Ca 2+ -binding proteins [68], as well as effects of plant hormones on signalling networks [69]. Kranner and Seal proposed [70] a triphasic stress model of seed redox control under salt stress, whereby the 'alarm' phase involves stress perception and transduction through the ROS-RNS-hormone signalling network, post-translational modification of macromolecules and an altered transcriptome so that the protection and repair machinery become activated and upregulated, respectively, in response to the perception of a stress and / or the initial damage caused. Under continuing stress (time or severity), the 'resistance' phase is reached when sufficient gene products required for protection and repair are produced to maintain viability. The resistance phase includes inducible protection (e.g. upregulated antioxidants that protect macromolecules from further damage), repair mechanisms (e.g. synthesis of DNA repair enzymes), and the elimination of redundant cells that are damaged beyond repair.
On the other hand, our understanding of seed tolerance or resistance to heavy metal exposure is far from complete and future research should be directed to achieve a better understanding of the mechanisms by which they act or interact with biomolecules and metabolic pathways during early and post-germination phases. The present work suggests that differential responses within different organs of the same seed may be due to differential mechanisms  that confer stress resistance. There might also be much interaction, with feedback loops between gene expression, transcription and translation as well as interconnections between the various biochemical pathways responsible for metal tolerance, such as those that define the redox hub comprising ROS, antioxidants and plant hormones. Once we have achieved a more complete understanding of the pathways that confer tolerance to salinity and drought, it may be possible to up-or down-regulate sets of genes until those required for salt tolerance, or more generally, stress tolerance, have been identified. Our approach has profiled profound biochemical changes associated with development of oxidative stress under environmental stress conditions. The data reported here provide novel insights that may lead to a broader understanding of molecular responses to Cu-induced stress in higher plants, and the resulting consequences for growth, development and enhanced agricultural productivity.