In this study, the metabolic adjustments performed by maize ( Zea mays L.) seminal roots exposed to 25 µM Cd 2+ or 25 µM Cu 2+ at pre-emergence are compared, focusing on the proteomic changes after metal exposure. Root width was increased, and root length was decreased after 72 h of metal treatment. Both metals induced H 2 O 2 accumulation and lipid peroxidation in the root tip. These changes were accompanied by increases in lipoxygenase activity and 4-hydroxy-2-nonenal content. NMR spectroscopy revealed that the abundance of 38 water-soluble metabolites was significantly modified by Cd and Cu exposure; this set of metabolites comprised carboxylic acids, amino acids, carbohydrates, and unidentified phenolic compounds. Linoleic acid content significantly decreased in Cu-treated samples. The total amount of proteins detected in maize root apexes was 2,171. Gene ontology enrichment analysis of the differentially accumulated proteins was performed to detect pathways probably affected by metal additions. Both metals altered redox homeostasis, up-regulated oxylipins biosynthetic process, and shifted metabolism towards the oxidative pentose-phosphate in the root apexes. However, the methionine salvage pathway appears as a key metabolic module only under Cd stress. The integrative analysis carried out in this study suggests that most molecular features behind the reprogramming of maize root tips to cope with cadmium and copper toxicity are common, but some are not.

Nitric oxide (NO) is a small gaseous molecule regarded as an important signaling compound. It induces several developmental and physiological responses in plants, ranging from germination and senescence to photosynthesis and redox homeostasis. It can interact with phytohormones, like abscisic acid and auxins, inhibit programmed cell death (PCD), and limit the detrimental effects of stress, including the mitigation of metal toxicity during heavy metal exposure. In this chapter, the current knowledge of the role of nitric oxide in plant response to metal stress is provided, with a special focus on cadmium stress. The involvement of NO in the redox homeostasis of plants, the crosstalk with phytohormones, and the posttranslational modifications produced by this molecule are discussed.

Cadmium (Cd) pollution of agricultural soils is a growing global concern. Plant growth restriction is the main visible symptom of Cd toxicity, and this metal may be particularly harmful to the preformed, seminal root during the pre-emergence stage. In the present study, we focused on Cd phytotoxicity in seminal root growth, nutrient composition, redox status, and hormone homeostasis during the pre-emergence stage of maize (Zea mays L) plants, distinguishing between the root apex and the remaining root tissue. After 72 h of metal exposure (50 and 100 µM CdCl2), root length and biomass, as well as Ca, Fe, Mg, and Mn contents, were diminished. A redox imbalance was evidenced by changes in peroxidase activities and the ascorbate–dehydroascorbate ratio decreased in both root parts. There were fewer carbonylated proteins in both root fractions after exposure to 50 µM Cd, compared to 100 µM Cd, which was related to increased 20S proteasome activities. Cd incremented ABA, IAA, and SA contents, but drastically reduced the biologically active gibberellin GA4 and the conjugate jasmonoyl-isoleucine (JA-Ile). We demonstrated that the whole root tissue is involved in the maize response to Cd stress, which entails redox and hormonal rearrangements, probably directed to widen the plant defense lines at the expense of root growth.

Nitrogen (N) is a central mineral nutrient essential for plant development and growth. It is usually scarcely found in soils, so the knowledge of the overall plant N metabolism deserves substantial attention. Polyamines (PAs) are N-containing low-molecular-weight compounds of polycationic nature involved in essential processes all throughout the life of plants whereas nitric oxide (NO) is a gaseous free radical involved in signalling cascades related to many physiological events. PAs and NO share signalling functions and interact with each other in several biological functions, mainly in stress responses. Biosynthesis pathways of PAs and NO are overlapped; PAs induce NO formation, but it is still not completely defined whether PAs act as substrates, cofactors, or signals for promoting NO synthesis and also, which are the mechanisms involved in NO regulation of PAs metabolism. Polyamine levels are of vital importance in the regulation of the network of N-metabolising pathways in plants, as they are components of the core of the overall N metabolism. In light of the importance of improving the efficiency of N uptake and distribution, it is time to elucidate the intricate relationship among N as a nutrient with PAs and NO as emerging signalling molecules. The close cooperation among these players in the whole N metabolism is an interesting target for the development of biotechnological tools for sustainable agriculture.

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.

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.

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.

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.

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.

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 β-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.

The numerous deleterious health effects upon exposure to toxic heavy metals in the environment are a matter of serious concern and a global issue. Cadmium is classified as a no-redox metal, but in biological systems, it is characterized by developing redox cell imbalance. It is well documented that in many plants, acute and chronic Cd exposure increases reactive oxygen and nitrogen species (ROS and RNS, respectively) in the cell. ROS and RNS could be acting as deleterious molecules inducing an oxidative/nitrosative injury that could result in growth inhibition, accelerated senescence, or cell death; or as essential components in the signaling cascade through plants' response to cope with metal stress. An understanding of the mechanisms by which plants perceive, transduce, and counterbalance Cd-induced oxidative or nitrosative stress signals to initiate acclimation or adaptive responses can be helpful in developing plants with increased tolerance to metal stress.

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.

Plants are exposed to environmental stress, in natural and agricultural conditions. Nitric oxide (NO), a small gaseous molecule which plays important roles in plants, has been involved in many physiological processes, and emerged as an important endogenous signaling molecule in the adaptation of plants to biotic and abiotic stress. NO is produced from a variety of enzymatic and non enzymatic sources, which are not yet fully understood. Also, NO and reactive nitrogen species (RNS) can produce posttranslational modifications affecting protein function. Nitrate reductase, a key enzyme in the nitrogen metabolism, is a proposed source of NO in plants which could be affected by posttranslational modifications. Thus, different pathways seem to be involved and can also regulate NO synthesis in the plant cell under physiological or stress conditions. However, how the levels of NO are reached in such time and place to fulfill its functions, are still puzzles to elucidate.

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.

In shaded wheat (Triticum aestivum L.) leaves, the suppression of blue radiation (BR) triggers senescence. This phenomenon is correlated to an increase in oxidative stress symptoms and a decrease of catalase (CAT) activity, among other traits. Previous data suggest that the radiation signal transduction pathway may involve changes in Ca2+ and H2O2 homeostasis. For better a understanding of the interaction among the spectral composition of radiation, Ca2+ availability, and the antioxidant metabolism in the regulation of shade-induced senescence, detached wheat leaves were placed in a growth chamber and exposed to either blue (B, high BR transmittance) and/or green (G, very low BR transmittance) Lee® filters in the absence or presence of 0.8 mM verapamil (a Ca2+ channels blocker), 4.0 mM EGTA (a Ca2+chelator), or 8.0 mM 3-amino-1,2,4-triazole (a CAT inhibitor). At defined time points, the leaf samples were analyzed for changes in chlorophyll content, specific activities of CAT, ascorbate peroxidase (APX), and guaiacol peroxidase (POX), CAT isozymes, and gene expression of CAT1, CAT2, and two senescence markers (TaSAG1 and TaSAG3). BR transmittance decreased the chlorophyll degradation rate and SAG genes expression either in leaves continuously exposed under the B filter, as well as in leaves previously exposed under the G filter. The effect of BR was associated with the maintenance of a high CAT (but not APX and POX) activity, and it was suppressed either in the presence of 3-AT or when Ca2+ availability was decreased. BR altered the CAT activity both at the transcriptional and at the posttranscriptional level. Nevertheless, different responses of CAT isozymes and CAT genes expression profiles to specific treatment combinations indicate that they differed in their regulatory pathways.

In this investigation we analyzed in detail the consequences of water deficit during the first 4 days of wheat development, focusing on root growth as affected by eventual changes in cell cycle regulation and oxidative processes. Root elongation decreased under water restriction in correlation with the intensity of this limitation, but the total number of cells between the quiescent center and the start of the rapid elongation zone in the root apex did not vary. Neither lipid peroxidation nor protein carbonylation increased in the roots of water-starved seedlings (ψ w : −0.6 MPa); accordingly, catalase activity increased, and transcript levels of cat2 gene were enhanced. Superoxide dismutase activity rose at day 2 and 3 and, unlike catalase, displayed quite similar levels on comparing roots and coleoptiles. Proline and total soluble carbohydrates increased in the roots of water-starved seedling. Total conductivity and osmolality were also augmented. No changes in the transcript levels of the markers related to G1-S transition phase of cell cycle could be detected. However, two expansin genes ( TaEXPB8 and TaEXPA5 ) were up-regulated in roots under water deficit. We conclude that wheat root elongation in water-deprived seedlings was simply hampered by lack of water income to cells. The enhanced expression of two root expansin genes is probably related to the eventual need of a quick cell wall expansion to allow the existing root cells to recover normal turgor, in case of sudden rewatering. Graphical Abstract Maintenance of redox balance, osmotic adjustment and modification of cell wall plasticity may be key points for wheat seedlings to overcome a water restriction during early growth.

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 Cu2+ 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 Cu2+ 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 Cd2+ and Cu2+ stress.

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 Cd²⁺ 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.