The Role of Alternative Oxidase in the Interplay between Nitric Oxide, Reactive Oxygen Species, and Ethylene in Tobacco (Nicotiana tabacum L.) Plants Incubated under Normoxic and Hypoxic Conditions.

The transgenic tobacco (Nicotiana tabacum L.) plants with the modified levels of alternative oxidase (AOX) were used to evaluate the physiological roles of AOX in regulating nitro-oxidative stress and metabolic changes after exposing plants to hypoxia for 6 h. Under normoxia, AOX expression resulted in the decrease of nitric oxide (NO) levels and of the rate of protein S-nitrosylation, while under hypoxia, AOX overexpressors exhibited higher NO and S-nitrosylation levels than knockdowns. AOX expression was essential in avoiding hypoxia-induced superoxide and H2O2 levels, and this was achieved via higher activities of catalase and glutathione reductase and the reduced expression of respiratory burst oxidase homolog (Rboh) in overexpressors as compared to knockdowns. The AOX overexpressing lines accumulated less pyruvate and exhibited the increased transcript and activity levels of pyruvate decarboxylase and alcohol dehydrogenase under hypoxia. This suggests that AOX contributes to the energy state of hypoxic tissues by stimulating the increase of pyruvate flow into fermentation pathways. Ethylene biosynthesis genes encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC oxidase, and ethylene-responsive factors (ERFs) were induced during hypoxia and correlated with AOX and NO levels. We conclude that AOX controls the interaction of NO, reactive oxygen species, and ethylene, triggering a coordinated downstream defensive response against hypoxia.

The Response of Cowpea (Vigna unguiculata) Plants to Three Abiotic Stresses Applied with Increasing Intensity: Hypoxia, Salinity, and Water Deficit.

Exposing plants to gradually increasing stress and to abiotic shock represents two different phenomena. The knowledge on plants' responses following gradually increasing stress is limited, as many of the studies are focused on abiotic shock responses. We aimed to investigate how cowpea (Vigna unguiculata (L.) Walp.) plants respond to three common agricultural abiotic stresses: hypoxia (applied with the increasing time of exposure to nitrogen gas), salinity (gradually increasing NaCl concentration), and water deficit (gradual decrease in water supply). We hypothesized that the cowpea plants would increase in tolerance to these three abiotic stresses when their intensities rose in a stepwise manner. Following two weeks of treatments, leaf and whole-plant fresh weights declined, soluble sugar levels in leaves decreased, and lipid peroxidation of leaves and roots and the levels of leaf electrolyte leakage increased. Polyphenol oxidase activity in both roots and leaves exhibited a marked increase as compared to catalase and peroxidase. Leaf flavonoid content decreased considerably after hypoxia, while it increased under water deficit treatment. NO emission rates after 3 h in the hypoxically treated plants were similar to the controls, while the other two treatments resulted in lower values of NO production, and these levels further decreased with time. The degree of these changes was dependent on the type of treatment, and the observed effects were more substantial in leaves than in roots. In summary, the responses of cowpea plants to abiotic stress depend on the type and the degree of stress applied and the plant organs.

Nitric Oxide Turnover Under Hypoxia Results in the Rapid Increased Expression of the Plastid-Localized Phosphorylated Pathway of Serine Biosynthesis.

The plant mitochondrial electron transport chain influences carbon and nitrogen metabolism under near anoxic conditions through its involvement in the phytoglobin-nitric oxide cycle, where the respiratory chain reduces nitrite to nitric oxide (NO), followed by NO conversion to nitrate by class 1 phytoglobin. Wild type (WT) and transgenic tobacco (Nicotiana tabacum L.) with differing amounts of alternative oxidase (AOX) were used to manipulate NO generation under hypoxia, and to examine whether this in turn influenced the gene expression of two stress-related amino acid biosynthetic pathways, the plastid-localized phosphorylated pathway of serine biosynthesis (PPSB), and the γ-aminobutyric acid (GABA) shunt. Under hypoxia, leaf NO emission rate was highest in AOX overexpressors and lowest in AOX knockdowns, with WT showing an intermediate rate. In turn, the rate of NO emission correlated with the degree to which amino acids accumulated. This amino acid accumulation was associated with the increased expression of the enzymes of the stress-related amino acid biosynthetic pathways. However, induction of the PPSB occurred much earlier than the GABA shunt. This work shows that high rates of NO turnover associate with rapid gene induction of the PPSB, establishing a clear link between this pathway and the maintenance of carbon, nitrogen and energy metabolism under hypoxia.

Transcriptional and Metabolic Changes Associated with Phytoglobin Expression during Germination of Barley Seeds.

To understand how the class 1 phytoglobin is involved in germination process via the modulation of the nitric oxide (NO) metabolism, we performed the analysis of physiological and molecular parameters in the embryos of transgenic barley (Hordeum vulgare L. cv Golden Promise) plants differing in expression levels of the phytoglobin (Pgb1) gene during the first 48 h of germination. Overexpression of Pgb1 resulted in a higher rate of germination, higher protein content and higher ATP/ADP ratios. This was accompanied by a lower rate of NO emission after radicle protrusion, as compared to the wild type and downregulating line, and a lower rate of S-nitrosylation of proteins in the first hours postimbibition. The rate of fermentation estimated by the expression and activity of alcohol dehydrogenase was significantly higher in the Pgb1 downregulating line, the same tendency was observed for nitrate reductase expression. The genes encoding succinate dehydrogenase and pyruvate dehydrogenase complex subunits were more actively expressed in embryos of the seeds overexpressing Pgb1. It is concluded that Pgb1 expression in embryo is essential for the maintenance of redox and energy balance before radicle protrusion, when seeds experience low internal oxygen concentration and exerts the effect on metabolism during the initial development of seedlings.

A comparative study of biotechnological approaches for producing valuable flavonoids in Prosopis farcta.

The callus and hairy root cultures of Prosopis farcta were established to develop effective strategies to enhance its valuable and medicinally important flavonoid compounds. For callus induction, the hypocotyl, cotyledon and shoot explants were subjected to different plant hormones, naphthalene acetic acid (NAA), benzylaminopurine (BAP), kinetin and dichlorophenoxyacetic acid (2,4-D). Greater callus induction was obtained from hypocotyl explants on MS medium containing 3.0 mg L-1 NAA + 2.0 mg L-1 BAP. With the addition of 0.5 mg L-1 asparagine to this medium, the maximum callus growth was achieved. Hairy root culture of P. farcta was performed using transformation of different explants with strains of Agrobacterium rhizogenes LBA9404, A4, AR15834. The AR15834 strain was more effective for hairy root induction where it caused hairy root formation on 59% of the infected cotyledon explants. We compared profiles of flavonoids isolated from seedling roots, hairy roots, and callus cultures of P. farcta. The colorimetric analysis showed that the content of total flavonoids of hairy roots was 1.54 and 2.52 times higher than in seedling roots and callus, respectively. The presence of flavonoids was verified by LC/MS in positive ion mode. The results showed that flavonoid composition was different in the roots and callus. Naringenin was the major constituent in callus, whereas resveratrol, quercetin and myricetin were the most abundant compounds found in hairy roots. The main objective of this research was to establish hairy roots in P. farcta to synthesize flavonoids at levels comparable to in vitro-grown roots. The present study also opens up a way to further improve the production of pharmaceutically valuable flavonoids and to produce desired metabolites using the hairy root culture system.

Favouring NO over H2O2 production will increase Pb tolerance in Prosopis farcta via altered primary metabolism.

Reactive oxygen species (ROS) and nitric oxide (NO) are known in triggering defense functions to detoxify heavy metal stresses. To investigate the relevance of ROS production, Pb treatment (400µM) alone and in combination with 400µM sodium ascorbate (Asc: as H2O2 scavenger) were given to hydroponically grown Prosopis farcta seedlings over a time course of 72h. Data presented here indicate that, the low extent of H2O2 due to scavenging by ascorbate, together with high level of NO improved Pb+Asc- treated Prosopis growth. Following the evoked potential of both the signals, significant increases in phenolic acids; caffeic, ferulic and salicylic acid were observed with Pb treatment; which are consistent with observed increase in lignin content and consequently with growth inhibition. In contrast, Pb+Asc treatment induced more flavonoids (quercetin, kaempferol, luteolin), diminished phenolic acids contents and also lignin. Elicited expression rate of phenylalanine ammonia-lyase gene (PAL) and also its enzymatic activity verified the induced phenylpropanoid metabolism by Pb and Pb+Asc treatments. In comparison with Pb stress, Asc+Pb application induced the high expression of arginine decarboxylase gene (ADC), in polyamines biosynthesis pathway, and conducted the N flow towards polyamines and γ-amino butyric acid (GABA). Examining the impact on enzyme activities, catalase, and guaiacol peroxidase; Pb+Asc reduced activity but this increased ascorbate peroxidase, and aconitase activity. Our observations are consistent with conditions favouring NO production and reduced H2O2 can improve Pb tolerance via wide-ranging effects on a primary metabolic network.

Modulation of Pb-induced stress in Prosopis shoots through an interconnected network of signaling molecules, phenolic compounds and amino acids.

Lead (Pb) is a hazardous heavy metal present in the environment which elicits oxidative stress in plants. To characterize the physiological and biochemical basis of Pb tolerance, Prosopis farcta seedlings were exposed to Hoagland's solutions at six different Pb concentrations (0, 80, 160, 320, 400 and 480 µM) for different periods of time. As expected, application of Pb significantly increased hydrogen peroxide (H2O2) content. In response, P. farcta deployed the antioxidative defence mechanisms with significantly higher activities of superoxide dismutase (SOD), enzymes related to H2O2 removal, and also the increases in proline as a solute marker of stress. Increases were observed in nitric oxide (NO) production which could also act in triggering defense functions to detoxify Pb. Enhanced phenylalanine ammonia-lyase (PAL) activity at early days of exposure to Pb was correlated with increases in phenolic compounds. Significant increases in phenolic acids and flavonoids; daidzein, vitexin, ferulic acid and salicylic acid were observed with Pb treatment. Furthermore, the stress effects were followed by changes in free amino acid content and composition. Aspartic acid and glycine content was increased but glutamic acid significantly decreased. It is likely that stress signal transduction by NO and H2O2 mediated defence responses to Pb by coordination of antioxidative system and metabolic pathways of phenylpropanoid and amino acids.