An isopentenyl transferase transgenic wheat isoline exhibits less seminal root growth impairment and a differential metabolite profile under Cd stress.

Cadmium is one of the most important contaminants and it induces severe plant growth restriction. In this study, we analyzed the metabolic changes associated with root growth restriction caused by cadmium in the early seminal root apex of wheat. Our study included two genotypes: the commercial variety ProINTA Federal (WT) and the PSARK ::IPT (IPT) line which exhibit high-grade yield performance under water deficit. Root tips of seedlings grown for 72 h without or with 10 µM CdCl2 (Cd-WT and Cd-IPT) were compared. Root length reduction was more severe in Cd-WT than Cd-IPT. Cd decreased superoxide dismutase activity in both lines and increased catalase activity only in the WT. In Cd-IPT, ascorbate and guaiacol peroxidase activities raised compared to Cd-WT. The hormonal homeostasis was altered by the metal, with significant decreases in abscisic acid, jasmonic acid, 12-oxophytodienoic acid, gibberellins GA20, and GA7 levels. Increases in flavonoids and phenylamides were also found. Root growth impairment was not associated with a decrease in expansin (EXP) transcripts. On the contrary, TaEXPB8 expression increased in the WT treated by Cd. Our findings suggest that the line expressing the PSARK ::IPT construction increased the homeostatic range to cope with Cd stress, which is visible by a lesser reduction of the root elongation compared to WT plants. The decline of root growth produced by Cd was associated with hormonal imbalance at the root apex level. We hypothesize that activation of phenolic secondary metabolism could enhance antioxidant defenses and contribute to cell wall reinforcement to deal with Cd toxicity.

Optimization of recombinant maize CDKA;1 and CycD6;1 production in Escherichia coli by response surface methodology.

The complex formed by the cyclin-dependent kinase A (CDKA) and cyclin D is responsible for the G1-S transition in the plant cell cycle. Maize (Zea mays L) CDKA; 1 and CycD6; 1 were cloned and expressed in E. coli. The present study describes the optimization of both proteins production using a statistical approach known as response surface methodology (RSM). The experimental design took into account the effects of four variables: optical density of the culture (OD600) before induction, isopropyl ß-d-1-thiogalactopyranoside (IPTG) concentration, post-induction temperature, and post-induction time. For each protein, a 24 full factorial central composite rotary design for these four independent variables (at five levels each) was employed to fit a polynomial model; which indicated that 30 experiments were required for this procedure. An optimization of CDKA; 1 and CycD6; 1 production levels in the soluble fraction was achieved. Protein conformation and stability were studied by circular dichroism and fluorescence spectroscopy. Finally, in vitro Cyc-CDK complex formation and its kinase activity were confirmed.

Early responses of maize seedlings to Cu stress include sharp decreases in gibberellins and jasmonates in the root apex.

Copper (Cu) interferes with numerous biological functions in plants, including plant growth, which is partly governed by plant hormones. In the present study, Cu stress effect on the roots of pre-emerging maize seedlings in terms of growth, nutrient composition, protein modifications, and root hormone homeostasis was investigated, focusing on possible metabolic differences between the root apex and the rest of the root tissues. Significant decreases in root length and root biomass after 72 h of Cu exposure (50 and 100 µM CuCl2), accompanied by reductions in Ca, Mg, and P root contents, were found. Cu also generated cell redox imbalance in both root tissues and revealed by altered enzymatic and non-enzymatic antioxidant defenses. Oxidative stress was evidenced by an increased protein carbonylation level in both tissues. Copper also induced protein ubiquitylation and SUMOylation and affected 20S proteasome peptidase activities in both tissues. Drastic reductions in ABA, IAA, JA (both free and conjugated), GA3, and GA4 levels in the root apex were detected under Cu stress. Our results show that Cu exposure generated oxidative damage and altered root hormonal homeostasis, mainly at the root apex, leading to a strong root growth inhibition. Severe protein post-translational modifications upon Cu exposure occurred in both tissues, suggesting that even when hormonal adjustments to cope with Cu stress occurred mainly at the root apex, the entire root is compromised in the protein turnover that seems to be necessary to trigger and/or to sustain defense mechanisms against Cu toxicity.

Tyr-nitration in maize CDKA;1 results in lower affinity for ATP binding.

Cyclin-dependent kinase A (CDKA) is a key component for cell cycle progression. The catalytic kinase activity depends on the protein's ability to form an active complex with cyclins and on phosphoregulatory mechanisms. Cell cycle arrest and plant growth impairment under abiotic stress have been linked to different molecular processes triggered by increased levels of reactive oxygen and nitrogen species (ROS and RNS). Among these, posttranslational modifications (PTMs) of key proteins such as CDKA;1 may be of significance. Herein, isolated maize embryo axes were subjected to sodium nitroprusside (SNP) as an inductor of nitrosative conditions to evaluate if CDKA;1 protein was a target for RNS. A high degree of protein nitration was detected; this included the specific Tyr-nitration of CDKA;1. Tyr15 and Tyr19, located at the ATP-binding site, were the selective targets for nitration according to both in silico analysis using the predictive software GPS-YNO2, and in vitro mass spectrometry studies of recombinant nitrated ZmCDKA;1. Spectrofluorometric measurements demonstrated a reduction of ZmCDKA;1-NO2 affinity for ATP. From these results, we conclude that Tyr nitration in CDKA;1 could act as an active modulator of cell cycle progression during redox stress.

Metabolic rearrangements in imbibed maize (Zea mays L) embryos in the presence of oxidative stressors.

Cadmium (Cd) is a metal known to generate oxidative stress in plants and may be particularly harmful during germination. Herein, the growth and metabolic rearrangements of maize embryo axes subjected during the imbibition stage to Cd ions and other two well-known oxidative stressors, methyl viologen (MV) and hydrogen peroxide (H2O2), were assessed for 48 h. Similar decreases in embryo's length were detected for all stressed axes up to 48 h of imbibition. By this time, treated embryos revealed greater accumulation of reactive oxygen species (ROS) and increased levels of carbonylated and ubiquitinated proteins. The proteolytic activities were intensely enhanced in the treated axes, particularly at 48 h of imbibition, and several antioxidant enzymes were induced in most cases. NMR spectroscopy followed by principal component analysis (PCA) and hierarchical cluster analysis (HCA) showed that a large proportion of polar metabolites, mainly amino acids and organic acids, were decreased under stress conditions, while carbohydrates were increased at 48 h of imbibition, with significant increases in glucose and raffinose for treated embryos relatively to controls. We demonstrated that maize embryo axes were capable of shifting their metabolism to improve their antioxidant defense system, at the expense of their growth. Under these adverse conditions, proteolysis seems to play a key role by providing free amino acids needed for the de novo synthesis of defense-related proteins.

Oxidation of proline from the cyclin-binding motif in maize CDKA;1 results in lower affinity with its cyclin regulatory subunit.

Cyclin dependent kinase A; 1 (CDKA; 1) is essential in G1/S transition of cell cycle and its oxidation has been implicated in cell cycle arrest during plant abiotic stress. In the present study, an evaluation at the molecular level was performed to find possible sites of protein oxidative modifications. In vivo studies demonstrated that carbonylation of maize CDKA,1 is associated with a decrease in complex formation with maize cyclin D (CycD). Control and in vitro oxidized recombinant CDKA; 1 were sequenced by mass spectrometry. Proline at the PSTAIRE cyclin-binding motif was identified as the most susceptible oxidation site by comparative analysis of the resulted peptides. The specific interaction between CDKA; 1 and CycD6; 1, measured by surface plasmon resonance (SPR), demonstrated that the affinity and the kinetic of the interaction depended on the reduced-oxidized state of the CDKA; 1. CDKA; 1 protein oxidative modification would be in part responsible for affecting cell cycle progression, and thus producing plant growth inhibition under oxidative stress.

Heavy metals effects on proteolytic system in sunflower leaves.

Plant proteolytic system includes proteases, mainly localized inside the organelles, and the ubiquitin-proteasome pathway in both, the cytoplasm and the nucleus. It was recently demonstrated that under severe Cd stress sunflower (Helianthus annuus L.) proteasome activity is reduced and this results in accumulation of oxidized proteins. In order to test if under other heavy metal stresses sunflower proteolytic system undergoes similar changes, an hydroponic experiment was carried out. Ten days old sunflower plants were transferred to hydroponic culture solutions devoid (control) or containing 100 microM of AlCl(3), CoCl(2), CuCl(2), CrCl(3), HgCl(2), NiCl(2), PbCl(2) or ZnCl(2) and analyzed for protein oxidative damage and proteolytic activities. After 4 days of metal treatment, only Co(2+), Cu(2+), Hg(2+), and Ni(2+) were found to increase carbonyl groups content. Except for Al(3+) and Zn(2+), all metals tested significantly reduced all proteasome activities (chymotrypsin-like, trypsin-like and PGPH) and acid and neutral proteases activities. The effect on basic proteases was more variable. Abundance of 20S protein after metal treatments was similar to that obtained for control samples. Co(2+), Cu(2+), Hg(2+), Ni(2+), Cr(3+), and Pb(2+) induced accumulation of ubiquitin conjugated proteins. It is concluded that heavy metal effects on proteolytic system cannot be generalized; however, impairment of proteasome functionality and decreased proteases activities seem to be a common feature involved in metal toxicity to plants.

20S proteasome and accumulation of oxidized and ubiquitinated proteins in maize leaves subjected to cadmium stress.

In order to examine the possible involvement of the 20S proteasome in degradation of oxidized proteins, the effects of different cadmium concentrations on its activities, protein abundance and oxidation level were studied using maize (Zea mays L.) leaf segments. The accumulation of carbonylated and ubiquitinated proteins was also investigated. Treatment with 50 microM CdCl(2) increased both trypsin- and PGPH-like activities of the 20S proteasome. The incremental changes in 20S proteasome activities were probably caused by an increased level of 20S proteasome oxidation, with this being responsible for degradation of the oxidized proteins. When leaf segments were treated with 100 microM CdCl(2), the chymotrysin- and trypsin-like activities of the 20S proteasome also decreased, with a concomitant increase in accumulation of carbonylated and ubiquitinated proteins. With both Cd(2+) concentrations, the abundance of the 20S proteasome protein remained similar to the control experiments. These results provide evidence for the involvement of this proteolytic system in cadmium-stressed plants.

Mechanism of CATA3 induction by cadmium in sunflower leaves.

One of the main antioxidant enzymes, catalase (CAT, EC 1.11.1.6), is capable of catalyzing the dismutation of H(2)O(2). This enzyme is involved in signal transduction pathway in plants, controlling the cellular level of this reactive oxygen species. Four different genes, CATA1-CATA4, were identified in Helianthus annuus L. cotyledons. Incubation of sunflower leaf discs with 300 and 500 microM CdCl(2) under light conditions increased CATA3 transcript level. However, it was not induced by Cd(2+) in etiolated plants. This Cd(2+)-induced increase was reverted by adding 10mM ascorbate. Treatments with 0.4 and 10 microM rose bengal (a generator of (1)O(2)) did not activate CATA3, but 10 microM methyl viologen (an enhancer of O(2)(-) production) and 10 mM H(2)O(2) increased its expression. In isolated chloroplasts, Cd(2+) and methyl viologen produced oxidation of the probe 2',7'-dichlorofluorescein diacetate indicating ROS formation. Besides, Cd(2+) treatment of leaf discs under light decreased CAT activity and increased carbonyl groups content, thus suggesting that enzyme inactivation could be due - in part - to a protein oxidation. These results indicate that light is involved in Cd(2+)-induced CATA3 enhancement, which leads to the synthesis of CAT isoforms less sensible to oxidation, and that chloroplast might be the main source of ROS responsible for this process.

Proteolytic system in sunflower (Helianthus annuus L.) leaves under cadmium stress.

The effect of oxidative stress induced by cadmium on growth parameters and on the balance between protein synthesis and degradation was studied in sunflower (Helianthus annuus L.) leaves. Plants were germinated for 10 days and then transferred to hydroponic medium devoid (control) or containing 100, 200 and 300µM CdCl2. Analyses were performed between days 0 and 4 of Cd-treatment. All Cd(2+) concentrations significantly reduced leaf area and, fresh and dry weight, but leaf relative water content only decreased with 200 and 300µM Cd(2+). Control and treated plants had similar soluble protein content and showed the same rate of soluble protein labeling under the assay conditions. Although protease activity increased with cadmium treatment, proteasome activity was significantly inhibited. Expression of 20S proteasome remained similar to controls in cadmium treated plants. Cadmium caused an increase in ubiquitin-conjugated proteins and carbonyl groups content of treated plants, compared to control values. Cadmium induced an increase in protease specific activity; nevertheless, this increase was not relevant enough to avoid accumulation of oxidized proteins. Oxidation of proteins is one of the most important effects of cadmium treatment. The results presented here provide evidence for the role of the proteolytic system in sunflower plants subjected to cadmium stress.

Coronatine Inhibits Stomatal Closure through Guard Cell-Specific Inhibition of NADPH Oxidase-Dependent ROS Production.

Microbes trigger stomatal closure through microbe-associated molecular patterns (MAMPs). The bacterial pathogen Pseudomonas syringae pv. tomato (Pst) synthesizes the polyketide toxin coronatine, which inhibits stomatal closure by MAMPs and by the hormone abscisic acid (ABA). The mechanism by which coronatine, a jasmonic acid-isoleucine analog, achieves this effect is not completely clear. Reactive oxygen species (ROS) are essential second messengers in stomatal immunity, therefore we investigated the possible effect of coronatine on their production. We found that coronatine inhibits NADPH oxidase-dependent ROS production induced by ABA, and by the flagellin-derived peptide flg22. This toxin also inhibited NADPH oxidase-dependent stomatal closure induced by darkness, however, it failed to prevent stomatal closure by exogenously applied H2O2 or by salicylic acid, which induces ROS production through peroxidases. Contrary to what was observed on stomata, coronatine did not affect the oxidative burst induced by flg22 in leaf disks. Additionally, we observed that in NADPH oxidase mutants atrbohd and atrbohd/f, as well as in guard cell ABA responsive but flg22 insensitive mutants mpk3, mpk6, npr1-3, and lecrk-VI.2-1, the inhibition of ABA stomatal responses by both coronatine and the NADPH oxidase inhibitor diphenylene iodonium was markedly reduced. Interestingly, coronatine still impaired ABA-induced ROS synthesis in mpk3, mpk6, npr1-3, and lecrk-VI.2-1, suggesting a possible feedback regulation of ROS on other guard cell ABA signaling elements in these mutants. Altogether our results show that inhibition of NADPH oxidase-dependent ROS synthesis in guard cells plays an important role during endophytic colonization by Pst through stomata.

Priming with NO controls redox state and prevents cadmium-induced general up-regulation of methionine sulfoxide reductase gene family in Arabidopsis.

In the present study we evaluated the pre-treatment (priming) of Arabidopsis thaliana plants with sodium nitroprusside (SNP), a NO-donor, as an interesting approach for improving plant tolerance to cadmium stress. We focused on the cell redox balance and on the methionine sulfoxide reductases (MSR) family as a key component of such response. MSR catalyse the reversible oxidation of MetSO residues back to Met. Five MSRA genes and nine MSRB genes have been identified in A. thaliana, coding for proteins with different subcellular locations. After treating 20 days-old A. thaliana (Col 0) plants with 100 µM CdCl2, increased protein carbonylation in leaf tissue, lower chlorophyll content and higher levels of reactive oxygen species (ROS) in chloroplasts were detected, together with increased accumulation of all MSR transcripts evaluated. Further analysis showed reduction in guaiacol peroxidase activity (GPX) and increased catalase (CAT) activity, with no effect on ascorbate peroxidase (APX) activity. Pre-exposition of plants to 100 µM SNP before cadmium treatment restored redox balance; this seems to be linked to a better performance of antioxidant defenses. Our results indicate that NO priming may be acting as a modulator of plant antioxidant system by interfering in oxidative responses and by preventing up-regulation of MSR genes caused by metal exposure.

Early response of wheat seminal roots growing under copper excess.

Growth reduction caused by copper excess during plant photoautotrophic metabolism has been widely investigated, but information regarding early responses of root apical meristem (RAM) to toxic concentrations of this metal at the initial heterotrophic stage is certainly scarce. We analysed some determinants of seminal root growth in developing wheat seedlings germinated in the presence of 1, 5 and 10 µM CuCl2, focussing on oxidative damage to cell membrane and to proteins, and investigated the expression patterns of some genes relevant to cell cycle progression and cell expansion. The proliferation zone of the RAM was shorter under 5 and 10 µM CuCl2. Cyclin D and CDKA levels remained unchanged in the root apexes of wheat seedlings grown under these Cu(2+) concentrations, but more carbonylated levels of both proteins and less ubiquitinated-cyclin D was detected under 10 µM CuCl2. Increased levels of ROS were revealed by fluorescent probes at this Cu(2+) dose, and severe cell membrane damage took place at 5 and 10 µM CuCl2. Several genes related to retinoblastome phosphorylation and therefore involved in the transition from G1 to S cell cycle stage were found to be downregulated at 10 µM CuCl2, while most expansin genes here analysed were upregulated, even at a non-toxic concentration of 1 µM. These results together with previous findings suggest that a "common" signal which involves oxidative posttranslational modifications of specific cell cycle proteins may be necessary to induce root growth arrest under Cd(2+) and Cu(2+) stress.

Oxidative post translational modifications of proteins related to cell cycle are involved in cadmium toxicity in wheat seedlings.

Abiotic stress is greatly associated with plant growth inhibition and redox cell imbalance. In the present work, we have investigated in which way oxidative posttranslational modifications (PTM) of proteins related to cell cycle may be implicated in post-germinative root growth reduction caused by cadmium, by methyl viologen (MV) and by hydrogen peroxide (H2O2) in wheat seedlings. Although cadmium is considered a redox inactive metal, reactive oxygen species were detected in the apex root of metal-treated seedlings. Oxidative stress hastened cells displacement from the cell division zone to elongation/differentiation zone, resulting in a shortened meristem. The number of cells in the proliferation zone was lower after MV, H2O2 and 10 µM Cd²âº treatments compared to control. All treatments increased protein carbonylation. Although no modification in total Ub-conjugated proteins was detected, oxidative treatments reduced cyclin D and CDKA protein ubiquitination, concomitantly with a decrease in expression of cyclin D/CDKA/Rb/E2F-regulated genes. We postulate that ROS and oxidative PTM could be part of a general mechanism, specifically affecting G1/S transition and progression through S phase. This would rapidly block cell cycle progression and would allow the cellular defence system to be activated.

The control of root growth by reactive oxygen species in Salix nigra Marsh. seedlings.

The production of reactive oxygen species (ROS) in specific regions of Salix seedlings roots seems essential for the normal growth of this organ. We examined the role of different ROS in the control of root development in Salix nigra seedlings, and explored possible mechanisms involved in the regulation of ROS generation and action. Root growth was not significantly affected by OH quenchers, while it was either partially or completely inhibited in the presence of H2O2 or O2·â» scavengers, respectively. O2·â» production was elevated in the root apex, particularly in the subapical meristem and protodermal zones. Apical O2·â» generation activity was correlated to a high level of either Cu/Zn superoxide dismutase protein as well as carbonylated proteins. While NADPH-oxidase (NOX) was probably the main source of O2·â» generation, the existence of other sources should not be discarded. O2·â» production was also high in root hairs during budding, but it markedly decreased when the hair began to actively elongate. Root hair formation increased in the presence of H2O2 scavengers, and was suppressed when H2O2 or peroxidase inhibitors were supplied. The negative effect of H2O2 was partially counteracted by a MAPKK inhibitor. Possible mechanisms of action of the different ROS in comparison with other plant model systems are discussed.

Sunflower cotyledons cope with copper stress by inducing catalase subunits less sensitive to oxidation.

Copper is an essential trace element for living organisms, in excess, can be toxic to the cell because of its capacity to generate reactive oxygen species (ROS). Catalase (CAT) catalyzes the dismutation of hydrogen peroxide into water and dioxygen and in plants it is located in peroxisomes and glyoxysomes. Different metals can induce changes in CAT activity, but the mechanism underlying its changes is unclear. After 4h of treatment with 5 and 10 µM CuCl(2) a decrease in the specific CAT activity was detected in sunflower cotyledons of post-germinative heterotrophic seedlings. At 8h of treatment, 5 µM Cu(2+) produced an induction of CAT activity while only a complete recovery to control values was observed for 10 µM Cu(2+) treated seedlings. These activity variations were not related to the level of CAT protein expression, but they did correlate with the oxidative state of the CAT protein. This indicates that the mechanism of CAT inactivation by Cu(2+) involves oxidation of the protein structure. The level of the mRNA of CATA3 and CATA4 increased with the presence of the metal after 4h of exposure. These CAT genes code for the synthesis of CAT subunits less sensitive to oxidation, which would prevent the copper-induced oxidative inactivation of CAT.

Microcin J25 triggers cytochrome c release through irreversible damage of mitochondrial proteins and lipids.

We previously showed that the antimicrobial peptide microcin J25 induced the over-production of reactive oxygen species with the concomitant release of cytochrome c from rat heart mitochondria via the opening of the mitochondrial permeability transition pore. Here, we were able to demonstrate that indeed, as a consequence of the oxidative burst, MccJ25 induces carbonylation of mitochondrial proteins, which may explain the irreversible inhibition of complex III and the partial inhibition of superoxide dismutase and catalase. Moreover, the peptide raised the levels of oxidized membrane lipids, which triggers the release of cytochrome c. From in silico analysis, we hypothesize that microcin would elicit these effects through interaction with heme c1 at mitochondrial complex III. On the other hand, under an excess of l-arginine, MccJ25 caused nitric oxide overproduction with no oxidative damage and a marked inhibition in oxygen consumption. Therefore, a beneficial anti-oxidative activity could be favored by the addition of l-arginine. Conversely, MccJ25 pro-oxidative-apoptotic effect can be unleashed in either an arginine-free medium or by suppressing the nitric oxide synthase activity.

Modifications in catalase activity and expression in developing sunflower seedlings under cadmium stress.

Catalase (CAT) dismutates the reactive oxygen species H2O2 into water and dioxygen and in plants; it is located in peroxisomes and glyoxysomes. In the present study, we investigated the effect of cadmium (a well-known oxidative stress inducer) on catalase in roots and cotyledons of developing sunflower seedlings, at 10 microM and 100 microM. Although germination was unaltered after 48 h of exposure to 100 microM Cd2+, root length was significantly reduced. CAT activity was also significantly reduced, but this activity was completely restored (10 microM treatment) or even enhanced (100 microM treatment) 24 h later. Although CAT protein abundance remained similar to control in roots and cotyledons of Cd-treated seedlings, cadmium produced CAT protein oxidation, indicating that the mechanism of CAT inactivation by Cd2+ involves oxidation of the protein structure. The transcripts of the four genes described for sunflower (CATA1 to CATA4) increased after cadmium treatment; CATA1 and CATA2 were the most overexpressed in cotyledon and root, respectively. The differential expression of catalase genes in sunflower seedlings under Cd stress might be related to the synthesis of CAT isoforms less sensitive to oxidation, which would prevent enzyme inactivation and H2O2 accumulation.

Effect of cadmium stress on nitrogen metabolism in nodules and roots of soybean plants.

The nitrogen metabolism of soybean (Glycine max L.) nodules and roots was studied in plants subjected to two different concentrations (50 and 200 µM) of CdCl2. Nitrogenase activity was decreased in nodules treated with 200 µM Cd2+. In 50 µM Cd2+-treated plants, NH4+ content showed similar values to controls in nodules, but increased by 55% in roots. However, after treatment with 200 µM Cd2+, NH4+ levels increased in both tissues. Glutamate (Glu) and protein contents remained unaltered in nodules treated with 50 µM Cd2+, while at the higher Cd2+ concentration both were decreased. Nevertheless, polyamine content was increased at the two Cd2+ concentrations. In roots, Glu, polyamine and protein levels were significantly diminished at 50 and 200 µM CdCl2. For nitrogen-assimilation enzymes, glutamate dehydrogenase activity was moderately increased in nodules and roots following the lower Cd2+ treatment, though at the higher Cd2+ concentration root enzyme activity returned to control levels. An impressive increase in enzyme activity was found in nodules. In roots, the glutamine synthetase / glutamate synthase pathway was decreased at the two Cd2+ concentrations, though in nodules it was diminished only at 200 µM Cd2+. No changes in protease activity were found in the two tissues treated with 50µMCd2+. However, at 200 µM Cd2+, nodule and root protease activities decreased and increased, respectively. These results suggest that, in general, treatment with Cd2+ affects nitrogen assimilation and metabolism to a greater extent in soybean roots than in nodules.

Effect of different metals on protease activity in sunflower cotyledons

Proteases are crucial for living cells and play a role in plant cell adaptation to environmental conditions. Oxidative stress produced oxidized proteins which are selectively degraded by proteases. To understand the role of proteolysis in response to metal stress, sunflower plants (a plant suitable for phytoremediation) were treated with 100 µM of CdCl2, CuCl2, AlCl3, CoCl2, PbCl2, CrCl3, NiCl2, HgCl2 or ZnCl2. Changes in protease activity, gelatinase profile and protein oxidation were examined in sunflower cotyledons. Our results indicate that this tissue has mainly acid proteases belonging to different classes. Although all metals (except Zn) increased protein oxidation (62, 57, 112, 74, 74, 68, 64 and 40 percent for Pb, Al, Ni, Cd, Hg, Co, Cr and Cu over the control), they altered proteolysis in different ways. Pb, Al and Ni treatment decreased protease activity 22, 28 and 30 percent respect to control while Cd and Hg increased this activity in 23 and 27 percent. In Zn, Cu and Co treatments protease activity remained similar to control treatment. These results indicate that different proteases are involved in plant defence against metal toxicity. However, the identification of specific oxidized proteins involved in this process and the metal effect on class specific proteases should provide greater information.