María D. Groppa

Polyamines and abiotic stress: recent advances

Summary.In this review we will concentrate in the results published the last years regarding the involvement of polyamines in the plant responses to abiotic stresses, most remarkably on salt and drought stress. We will also turn to other types of abiotic stresses, less studied in relation to polyamine metabolism, such as mineral deficiencies, chilling, wounding, heavy metals, UV, ozone and paraquat, where polyamine metabolism is also modified.There is a great amount of data demonstrating that under many types of abiotic stresses, an accumulation of the three main polyamines putrescine, spermidine and spermine does occur. However, there are still many doubts concerning the role that polyamines play in stress tolerance. Several environmental challenges (osmotic stress, salinity, ozone, UV) are shown to induce ADC activity more than ODC. The rise in Put is mainly attributed to the increase in ADC activity as a consequence of the activation of ADC genes and their mRNA levels. On the other hand, free radicals are now accepted as important mediators of tissue injury and cell death. The polycationic nature of polyamines, positively charged at physiological pH, has attracted the attention of researchers and has led to the hypothesis that polyamines could affect physiological systems by binding to anionic sites, such as those associated with nucleic acids and membrane phospholipids. These amines, involved with the control of numerous cellular functions, including free radical scavenger and antioxidant activity, have been found to confer protection from abiotic stresses but their mode of action is not fully understood yet. In this review, we will also summarize information about the involvement of polyamines as antioxidants against the potential abiotic stress-derived oxidative damage.

Polyamines as protectors against cadmium or copper-induced oxidative damage in sunflower leaf discs

Abstract Polyamines antioxidant properties were studied in sunflower leaf discs under cadmium and copper induced oxidative stress. Both metals induced oxidative damage in the studied tissue, as evidenced by an increment in lipid peroxidation and a decrease in antioxidant enzymes ascorbate peroxidase (APOX), glutathione reductase (GR) and superoxide dismutase (SOD) activities. Polyamines concentrations were differently affected according to the metal used. Putrescine (Put) was reduced 52% by Cd 2+ and 39% by Cu 2+ , spermidine (Spd) was decreased to 21 and 59% of the control by Cd 2+ and Cu 2+ , respectively and spermine (Spm) content was not modified by any of the metals. Pretreatment with exogenously added polyamines (1 mM) showed that Spm reverted the effect of Cd 2+ and Cu 2+ on lipid peroxidation almost to control values. Neither Spm nor Spd recovered the metal-reduced APOX activity. Besides, both polyamines affected this parameter by themselves. GR activity was completely restored by Spm or Spd and only Spm was effective in reverting copper-reduced SOD activity. This work suggests that polyamines are undoubtedly related to the protection against metal-induced oxidative stress, but more research is necessary to elucidate the precise role they played as antioxidants.

Nitric oxide, polyamines and Cd-induced phytotoxicity in wheat roots.

To further explore the biochemical basis of Cd toxicity in developing wheat seedlings, we studied the possible role of nitric oxide (NO) and polyamines as signaling molecules involved in metal-induced root growth inhibition. When used at 0.1 mM, sodium nitroprusside, a NO-releasing compound, inhibited root growth to a similar extent as Cd and enhanced the polyamine contents as Cd also did. Putrescine and spermidine treatments caused significant decreases in root growth with spermine giving the greatest level of inhibition (77% reduction). The simultaneous addition of Cd and inhibitors of putrescine biosynthesis (DFMA and DFMO) prevented increases in putrescine levels but did not restore normal root growth. NO content, as evidenced by the fluorescent probe DAF-FM diacetate, was found to be significantly increased in the roots of both Cd and polyamine treated plants, especially in those exposed to spermine. The effect was specific for NO since the NO scavenger cPTIO almost suppressed the fluorescent signal. Concerning the oxidative status of the root system, only Cd and spermine enhanced lipid peroxidation in roots. At the same time, all treatments led to a significant increase in levels of the non-enzymatic antioxidant defense glutathione. Our results strongly suggest that Cd and spermine treatments induce NO formation in wheat roots which, in turn, is involved in root growth inhibition.

Polyamine metabolism in sunflower and wheat leaf discs under cadmium or copper stress

Abstract The influence of Cd 2+ or Cu 2+ on polyamine (Pa) metabolism and ethylene evolution was studied in sunflower and wheat leaf-discs. Both metals increased putrescine (Put) content in wheat leaves 280 and 90%, respectively, but reduced this Pa in sunflower leaves by about 50% of the control values. Spermidine content was diminished in cadmium and copper sunflower treated leaves (41 and 79%, respectively) while spermine was reduced by both metals in wheat (around 40%). In sunflower leaves, both metals greatly reduced arginine decarboxylase (ADC) activity, while ornithine decarboxylase (ODC) was only decreased by cadmium. However, in wheat leaves, cadmium increased ADC and ODC activities (210 and 2200% over the control, respectively), and Cu 2+ raised ODC activity fivefold respect to the control, with the resulting increase in Put concentration. Moreover, the decreased diamine oxidase activity observed with both metals in wheat leaves might contribute to the high Put level observed. Cadmium and copper increased ethylene production sixfold in wheat leaves, while in sunflower leaves, only copper produced a great increment in the hormone (300% over the control).

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.

Polyamine metabolism in sunflower plants under long-term cadmium or copper stress

Summary.The effect of different doses of cadmium and copper was studied in relation to growth and polyamine (Pas) metabolism in shoots of sunflower plants. Cadmium accumulated to higher levels than copper and shoot length was reduced by 0.5 and 1 mM Cd, but only by 1 mM Cu. At 1 mM of Cd or Cu, Put content increased 270% and 160% with Cd2+ and Cu2+, respectively. Spermidine (Spd) was modified only by 1 mM Cd, while spermine (Spm) declined after seeds germinated, increasing thereafter but only with 1 mM Cd or Cu (273% over the controls for Cd and 230% for Cu at day 16). Both ADC and ODC activities were increased by 1 mM Cd, whereas 1 mM Cu enhanced ADC activity, but reduced ODC activity at every concentration used. The role of Pas as markers of Cd or Cu toxicity is discussed.

Cadmium modulates NADPH oxidase activity and expression in sunflower leaves

The production of reactive oxygen species (ROS) and the ways by which ROS are generated are very important facts related to heavy metal toxicity in plants. In this work, superoxide anion (O2·−) generation diminished in cadmium treated sunflower (Helianthus annuus L.) leaf discs, and this reduction was time and Cd-concentration dependent. In line with these findings, we observed that NADPH-dependent oxidase activity was significantly inhibited by 0.1 and 0.5 mM Cd2+ treatments and the expression of the NADPH oxidase putative gene related to O2·− synthesis in sunflower leaves was 83 % inhibited by 0.1 mM CdCl2 and almost completely depleted by 0.5 mM CdCl2.

Effect of co-inoculation with Bradyrhizobium japonicum and Azospirillum brasilense on soybean plants

Abstract In order to assess the effects of co-inoculation with both Bradyrhizobium japonicum and Azospirillum brasilense on soybean plants, a pot experiment under growth controlled conditions was carried out. Four-week-old plants were harvested and analyzed for shoot, root and nodule dry weight, nitrogen content, nodule number and distribution, nodule protein and leghaemoglobin content, and nitrogenase and glutamine synthetase activities. Nodule number and nodule dry weight were not affected by dual inoculation; however, co-inoculated plants showed a significantly higher proportion of nodules attached to the main root and located in the upper 3 cm of the root system. Although no significant difference in total dry matter production could be detected, nitrogen content of dual inoculated plants was significantly increased (23 % over B. japonicum-single. inoculated plants). Accordingly, a strong stimulation of acetylene reduction activity was observed for this treatment. Likewise, leghaemoglobin content significantly increased (39 %). These results suggest that co-inoculation with B. japonicum and A. brasilense leads to an increased number of the most active nodules, therefore, to a greater nitrogen fixation and assimilation. In addition, an enhancement of nodule effectiveness due to A. brasilense presence in the root system is suggested.

Wheat seeds harbour bacterial endophytes with potential as plant growth promoters and biocontrol agents of Fusarium graminearum

The role of endophytic communities of seeds is still poorly characterised. The purpose of this work was to survey the presence of bacterial endophytes in the seeds of a commercial wheat cultivar widely sown in Argentina and to look for plant growth promotion features and biocontrol abilities against Fusarium graminearum among them. Six isolates were obtained from wheat seeds following a culture-dependent protocol. Four isolates were assignated to Paenibacillus genus according to their 16S rRNA sequencing. The only gammaproteobacteria isolated, presumably an Enterobactereaceae of Pantoea genus, was particularly active as IAA and siderophore producer, and also solubilised phosphate and was the only one that grew on N-free medium. Several of these isolates demonstrated ability to restrain F. graminearum growth on dual culture and in a bioassay using barley and wheat kernels. An outstanding ability to form biofilm on an inert surface was corroborated for those Paenibacillus which displayed greater biocontrol of F. graminearum, and the inoculation with one of these isolates in combination with the Pantoea isolate resulted in greater chlorophyll content in barley seedlings. Our results show a significant ecological potential of some components of the wheat seed endophytic community.

In vitro PGPR properties and osmotic tolerance of different Azospirillum native strains and their effects on growth of maize under drought stress

Osmotic variations in the soil can affect bacterial growth diminishing the number of inoculated bacteria. In a scenario of water deficit having tolerant bacteria would be beneficial to achieve a better response of the plant to stress. Thus, selection of more resistant bacteria could be useful to design new inoculants to be used in arid zones. In this sense, a group of Azospirillum isolates deposited in INTA collection was characterized in order to select strains tolerant to osmotic stress. The results obtained demonstrated that Az19 strain has similar in vitro PGPR characteristics to Az39, the most used strain in Argentina for inoculants industries, with the advantage of a better tolerance to osmotic and salt stress. Inoculation of maize plants with this strain resulted in a better response against water deficit compared to Az39 strain, encouraging us to further study the behavior of this strain in greenhouse and field trials in view of developing new inoculants suitable for areas with water deficit.