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.

Effects of polyamines on cadmium- and copper-mediated alterations in wheat (Triticum aestivum L) and sunflower (Helianthus annuus L) seedling membrane fluidity.

We investigated if wheat (Wh) and sunflower (Sf) plants watering with 1 mM CdCl2 or CuCl2 for 5-15 d during germination and seedling altered membrane fluidity (MF) of their leaves and roots, and if plant pre-treatment with the polyamines (PAs) putrescine (Put), spermidine (Spd) or spermine (Spm) prevented those alterations. Cd impaired Wh and Sf growth, while Cu only affected Sf growth. Cu and Cd increased MF of leaves of both plant species, while Cd decreased MF of Sf roots. Plant treatment for 15 d with 0.1 mM Put, Spd or Spm did not affect plant growth and had opposed effects on the MF of both plants. Finally, Wh and Sf were pre-treated with PAs for either 5 or 10 days followed by metal treatment until day 15. While Put did not affect membrane MF, Spd and Spm decreased it between 5 and 10 d of plant treatment. Together, experimental results demonstrate that during plant development (a) Cd and Cu have noxious effects on plants membrane biophysical properties that could be partially responsible of their toxicity, and (b) this deleterious effect could be only partially prevented by plant pretreatment with the PAs.

Response to saline stress and aquaporin expression in Azospirillum-inoculated barley seedlings.

The ability of two strains of Azospirillum brasilense to mitigate NaCl stress in barley plants was evaluated. Barley seedlings were inoculated and subjected to 200 mM NaCl for 18 days. Several days after NaCl treatment, a significant decline in biomass as well as in height was observed in uninoculated plants. However, smaller reductions in biomass and height were detected in plants inoculated with strain Az39. All the stressed plants showed significantly higher Na(+) but lower K(+) contents in their shoots. The growth rate of uninoculated plants was adversely affected by saline treatment, which was associated with higher putrescine content and lower levels of HvPIP2;1 transcripts in the roots. Azospirillum inoculation triggered the transcription of this gene. Our results suggest that barley plants inoculated with A. brasilense may be better prepared to thrive under saline conditions. To our knowledge, this is the first report showing an effect of Azospirillum inoculation on the expression of PIP2;1, a gene involved in the synthesis of root water channels.

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.

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.

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.

Inhibition of root growth and polyamine metabolism in sunflower (Helianthus annuus) seedlings under cadmium and copper stress.

Although sunflower is usually regarded as a highly tolerant crop, impairment of root growth at initial stages of plant development may result in poor crop establishment and higher susceptibility to pathogen attack. In order to evaluate if Cd2+ and Cu2+ may impact on sunflower germination and initial root development, a pot experiment under controlled conditions was carried out. Possible involvement of polyamine metabolism in sunflower response to these stressors was also investigated. Although Cd2+ and Cu2+ treatments affect neither seed germination nor radical emergence, sunflower seedlings grown in the presence of these heavy metals showed significant inhibition of root growth, being this inhibition greater for Cd2+. Both metals caused significant increases in proline contents at the highest concentrations tested (0.5 and 1 mM), and these increments were more pronounced for Cd2+ treatments, especially between days 3 and 10. Metals also increased putrescine (Put) contents at all concentrations assayed from the seventh day onward, causing no variations on this polyamine time-course pattern. Spermine and spermidine contents, however, were increased only by 1 mM Cd2+. Arginine decarboxylase seems to have been the enzyme responsible for Put increases under both metal treatments. This work demonstrates that initial root growth of sunflower seedlings may be significantly impaired in Cd2+ or Cu2+ contaminated soils. It also shows that polyamines are key biological compounds, which are probably involved in signaling pathways triggered under stress environmental conditions.

Polyamines and heavy metal stress: the antioxidant behavior of spermine in cadmium- and copper-treated wheat leaves.

Polyamine metabolism, as well as spermine (Spm) antioxidant properties, were studied in wheat leaves under Cd2+ or Cu2+ stress. The oxidative damage produced by both metals was evidenced by an increased of thiobarbituric acid reactive substances (TBARS) and a significant decrease in glutathione under both metal treatments. Ascorbate peroxidase (APOX) and glutathione reductase (GR) activities were reduced by both metals to values ranging from 30% to 64% of the control values. Conversely, copper produced a raise in superoxide dismutase activity. The high putrescine (Put) content detected under Cd2+ stress (282% over the control) was induced by the increased activity of both enzymes involved in Put biosynthesis, arginine decarboxylase (ADC) and ornithine decarboxylase (ODC). However, only ODC activity was increased in wheat leaves subjected to Cu2+ stress, leading to a lower Put rise (89% over the controls). Spermidine (Spd) content was not affected by metal treatments, while Spm was significantly reduced. Pretreatment with Spm completely reverted the metals-induced TBARS increase whereas metals-dependent H2O2 deposition on leaf segments (revealed using diaminobenzidine), was considerably reduced in Spm pretreated leaf segments. This polyamine failed to reverse the depletion in APOX activity and glutathione (GSH) content produced by Cd2+ and Cu2+, although it showed an efficient antioxidant behavior in the restoration of GR activity to control values. These results suggest that Spm could be exerting a certain antioxidant function by protecting the tissues from the metals-induced oxidative damage, though this effect was not enough to completely avoid Cd2+ and Cu2+ effect on certain antioxidant enzymes, though the precise mechanism of protection still needs to be elucidated.