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

https://doi.org/10.1016/j.ecoenv.2017.04.036Get rights and content

Highlights

  • Accumulation of free phenolic compounds in Prosopis was related to its growth inhibition.

  • Exogenous lead and ascorbic acid reduced phenolic acids and improved growth.

  • Accumulation of excess total amino acid is an important adaptive response in plants to stresses.

  • Different amounts of formation of NO and H2O2 can be channeled into specific pathways: survival and growth in conflicting demands.

Abstract

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 (400 µM) alone and in combination with 400 µM sodium ascorbate (Asc: as H2O2 scavenger) were given to hydroponically grown Prosopis farcta seedlings over a time course of 72 h. 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.

Introduction

Lead (Pb), one of the most abundant globally distributed toxic elements, posing a significant risk to the health of humans, animals, and plants. At the whole-plant level, high concentration of Pb causes the disruption of physiological and biochemical processes like a decrease in photosynthesis, altered uptake of essential elements, inhibition of growth, lower biomass and yields (Ali et al., 2014, Arias et al., 2010). At the molecular level, Pb changes cell membrane permeability, reacts with active groups of different enzymes (for example, haem groups), reacts with phosphate groups of ADP or ATP (Pourrut et al., 2011). These results in negative effects are associated with oxidative damage to plant cell due to a compromised antioxidant defense machinery and the production of reactive oxygen species (ROS) (Verma and Dubey, 2003). However, despite their potential for causing harmful oxidations, it is now well established that ROS; and most often H2O2, may also function as signaling molecule (Zafari et al., 2016, Ali et al., 2014).

To cope with oxidative stress, plants have evolved two protective enzymatic and non-enzymatic mechanisms to detoxify ROS species. The former includes catalase (CAT: E.C.1.11.1.6.), ascorbate peroxidase (APX: E.C.1.11.1.11), guaiacol peroxidase (GPX: E.C.1.11.1.7) and the latter involves ascorbate and glutathione (Gill and Tuteja, 2010); which work in concert to detoxify ROS. The low-molecular-weight antioxidant ascorbate functions as redox buffer that reduces ROS. It is the well-known molecule in the detoxification of H2O2, particularly as a substrate of APX, and is fundamental component of the ascorbate-glutathione cycle, which is present in most cellular compartments (Smirnoff and Wheeler, 2000). Crucially, ascorbate can also acts as a metabolic interface to modulate the appropriate induction of acclimation responses (Foyer and Noctor, 2005).

ROS and potentially ascorbate contribute to mechanisms that allow plants to withstand abiotic stresses that could affect their vigour and survival (Mittler, 2017, Huang et al., 2010). In response to metals exposure, plants accumulate different metabolites to concentrations in the millimolar range, particularly phenolic and nitrogenous compounds such as amino acids and polyamines (Sharma and Dietz, 2006). Plant phenolics contribute to ROS quenching and are considered as parts of defensive mechanism (Dǔcić et al., 2008). Crucially phenylalanine ammonia-lyase (PAL) gene expression is inducible by ROS (Lin et al., 2005), and in heavy metal-treated plants leads to phenolic acid accumulation (Kováčik et al., 2009). Also, the accumulation of some amino acids such as proline may help in ameliorating of negative consequences of metal toxicity (Kováčik et al., 2010). These solutes are also sources of carbon and nitrogen during environmental challenges (Dubay and Pessarakli, 1995).

Polyamines including putrescine (Put), spermidine (Spd) and spermine (Spm) influence various processes in controlling plant growth and development. Due to the presence of positively charged groups, the interaction of polyamines with proteins, nucleic acids, membrane phospholipids, and cell wall constituents can activate or stabilize these molecules. As such polyamines contribute significantly in enhancing plant defense strategies in response to abiotic stresses including heavy metal (Groppa and Benavides, 2008).

Along with H2O2, the free radical NO has gained special interest in plant signaling pathways controlling processes that range from biotic and abiotic stress responses to growth and development (Mur et al., 2012). Numerous studies have reported NO and H2O2 induce profound changes in the expression, enzymes activities and metabolite levels in phenylpropanoid and nitrogen pathways employed as tolerance mechanisms (Gao et al., 2009, Iqbal et al., 2014). Similarly, we have recently shown that NO acts as a signal molecule mediating Pb-induced stress tolerance in Prosopis farcta; a perennial trees/shrub species well known for their resistance to heavy metals (Zafari et al., 2016). However, it is likely that the Prosopis response is not triggered by NO alone but is the result of a cross-talk between different signals and metabolic pathways, especially ROS-dependent ones. This current study applied ascorbic acid as a H2O2 scavenger to establish that NO and H2O2 interact to regulate metabolome changes in Prosopis conducting to Pb tolerance strategies.

Section snippets

Plant culture and treatments

Prosopis farcta L. seeds were scarified with 98% sulphuric acid and sterilized in a solution of sodium hypochlorite (2% w/v), and then thoroughly rinsed in distilled water. Seeds were incubated at 25 °C for 3 days to germinate. Germinated seeds were then transferred into plastic containers with 2.5 dm3 of half strength Hoagland nutrient solution (pH 6). Plants were kept at 27 (light) /22 °C (dark) with a 16 h light photoperiod (200 µmol m−2 s−1) and 60–80% air humidity. After 21 days, uniform

Pb and Asc contents and plant growth

To provide further insight into the mechanisms of ROS mediated tolerance against Pb in Prosopis farcta we examined the impact of co-application of the ROS scavenger. Fig. 1a shows how feeding Prospois with 400 µM Pb increased absorbed Pb content (68.5 mg/kg) but the Pb content in the Pb+Asc-treated plants was 32% lower. To confirm the effect of exogenous Asc application its endogenous content in different samples was measured. Results showed that the ascorbate levels in response to Pb application

Discussion

ROS are produced as inevitable by-products of several metabolic pathways. In order to avoid ROS toxicity, plants are provided with a flexible set of enzymes and metabolites involved in ROS catabolism, which frequently acts at ROS generation sites (Mittler et al., 2004). Despite much metabolic energy being consumed on ROS scavenging by plant cells, ROS are also actively generated by cellular metabolic processes linked to optimal growth conditions. This aligns with growing evidence suggests that

Author contribution

This research paper was accomplished with the collaboration of all authors. Somaieh Zafari performed the experiments, analyzed and interpreted data and wrote the manuscript. Mohsen Sharifi designed and supervised the study. Luis A. J. Mur helped to evaluate and edit the manuscript. Najme Ahmadian Chashmi was the study advisor.

Acknowledgments

The authors greatly appreciate Tarbiat Modares University for supporting this research.

References (75)

  • A.U. Igamberdiev et al.

    Regulation of NAD- and NADP-dependent isocitrate dehydrogenases by reduction levels of pyridine nucleotides in mitochondria and cytosol of pea leaves

    Biochim. Et. Biophys. Acta

    (2003)
  • N. Iqbal et al.

    A new perspective of phytohormones in salinity tolerance: regulation of proline metabolism

    Environ. Exp. Bot.

    (2014)
  • J. Kováčik et al.

    Nitric oxide signals ROS scavenger-mediated enhancement of PAL activity in nitrogen-deficient Matricaria chamomilla roots: side effects of scavengers

    Free Radic. Biol. Med.

    (2009)
  • J. Kováčik et al.

    Effect of copper and salicylic acid on phenolic metabolites and free amino acids in Scenedesmus quadricauda (Chlorophyceae)

    Plant Sci.

    (2010)
  • J.S. Lin et al.

    Doubled CO could improve the drought tolerance better in sensitive cultivars than in tolerant cultivars in spring wheat

    Plant Sci.

    (2002)
  • W. Lin et al.

    Hydrogen peroxide mediates defence responses induced by chitosans of different molecular weights in rice

    J. Plant Physiol.

    (2005)
  • H.F. Liu et al.

    Relationship between osmotic stress and the levels of free, conjugated and bound polyamines in leaves of wheat seedlings

    Plant Sci.

    (2004)
  • R. Mittler

    ROS are good

    Trends Plant Sci.

    (2017)
  • R. Mittler et al.

    Reactive oxygen gene network of plants

    Trends Plant Sci.

    (2004)
  • R.W. Owen et al.

    Isolation, structure elucidation and antioxidant potential of the major phenolic and flavonoid compounds in brined olive drupes

    Food Chem. Toxicol.

    (2003)
  • E. Racker

    Spectrophotometric measurements of the enzymatic formation of fumaric and cis-aconitic acids

    Biochim. Et. Biophys. Acta

    (1950)
  • V. Rai et al.

    Effect of chromium accumulation on photosynthetic pigments, oxidative stress defence system, nitrate reduction, proline level and eugenol content of Ocimum tenuiflorum L

    Plant Sci.

    (2004)
  • A.I. Sannazzaro et al.

    Modulation of polyamine balance in Lotus glaber by salinity and arbuscular mycorrhiza

    Plant Physiol. Biochem.

    (2007)
  • M. Seki et al.

    Regulatory metabolic networks in drought stress responses

    Curr. Opin. Plant Biol.

    (2007)
  • P. Torrigiani et al.

    Improved method for polyamine determination in TMV a rod-shaped virus

    J. Virol. Methods

    (1995)
  • V. Velikova et al.

    Oxidative stress and some antioxidant systems in acid rain-treated bean plants

    Plant Sci.

    (2000)
  • S. Verma et al.

    Lead toxicity induces lipid peroxidation and alters the activities of antioxidant enzymes in growing rice plants

    Plant Sci.

    (2003)
  • S. Zafari et al.

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

    Plant Physiol. Biochem

    (2016)
  • X. Zhang et al.

    Roles of hydroxyproline-rich glycoproteins in the pollen tube and style cell growth of tobacco (Nicotiana tabacum L.)

    J. Plant Physiol.

    (2014)
  • N. Ahmadian Chashmi et al.

    Lignan enhancement in hairy root cultures of Linum album using coniferaldehyde and methylenedioxycinnamic acid

    Prep. Biochem. Biotechnol.

    (2016)
  • T. Ahmed Khan et al.

    A review of ascorbic acid potentialities against oxidative stress induced in plants

    J. Agrobiol.

    (2011)
  • R. Alcázar et al.

    Involvement of polyamines in plant response to abiotic stress

    Biotechnol. Lett.

    (2006)
  • R. Angelovici et al.

    Deciphering transcriptional and metabolic networks associated with lysine metabolism during Arabidopsis seed development

    Plant Physiol.

    (2009)
  • M. Babar Ali et al.

    Phenolics metabolism and lignin synthesis in root suspension cultures of Panax ginseng in response to copper stress

    Plant Sci.

    (2006)
  • L.S. Bates et al.

    Rapid determination of free proline for water-stress studies

    Plant Soil

    (1973)
  • M. Biermann et al.

    Simultaneous analysis of the non-canonical amino acids norleucine and norvaline in biopharmaceutical-related fermentation processes by a new ultra-high performance liquid chromatography approach

    Amino Acids

    (2013)
  • I. Cakmak et al.

    Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves

    Plant Physiol.

    (1992)
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