Skip to main content

Hemoglobin as a probe for estimation of nitric oxide emission from plant tissues



Plant roots contribute significant amount of nitric oxide (NO) in the rhizosphere as a component of NO in the ecosystem. Various pharmacological investigations on NO research in plants seek to quench endogenous NO by using externally applied NO quenchers, mainly 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) and its more soluble form-carboxy-PTIO (cPTIO). Owing to serious limitations in its application cPTIO is no more a desired compound for such applications.


Present work highlights the significance of using hemoglobin in the bathing solution to not only release endogenous NO from plant tissue but also to quench it in a concentration-dependent manner.


The protocol further demonstrates the diffusibility of NO from intracellular locations in presence of externally provided hemoglobin. The proposed method can have widespread applications as a substitute to debatable and currently used cPTIO as a NO scavenger.


Plants constitute an important source of biological NO emission in the terrestrial ecosystem [1, 2]. Following the first observation of NO emission from herbicide-treated soybean leaves into the atmosphere [3], several studies have focused on NO emissions from detached plant tissues, cell suspensions and mitochondria [2, 4,5,6,7].

NO is a gaseous, lipophilic biomolecule which acts as a free radical with ability to diffuse across cell membranes, through the cytoplasm and migrate intracellularly as well as from cell to cell across the apoplast. It diffuses at a rate of 50 μm s−1. Its solubility is 1.9 mM in aqueous solutions at 1 atm pressure. Half-life of NO in biological systems is reasonably short, less that 10 s. The rapid movement and removal of cellular NO, makes it an ideal signaling molecule for cell to cell communication in plant tissues both in normal growth conditions and under stress [3, 8,9,10,11,12]. It is a versatile molecule that can migrate and act concurrently in different cellular compartments and in opposite directions. NO is biosynthesized in plants via multiple routes which are broadly classified as reductive and oxidative pathways. It is produced through both enzymatically in plastids, mitochondria, chloroplasts, and non-enzymatically in the apoplast [6, 13,14,15,16,17,18,19].

Pharmacological investigations on the modulation of plant growth and development by NO routinely employ PTIO and its more soluble form-cPTIO as a means to quench tissue NO. Of late, it has been reported that cPTIO usage as a NO scavenger exhibits duality in its action [20]. Depending on concentration, cPTIO can, at times, even contribute to further NO production, rather than serving as a NO quencher. cPTIO oxidizes NO by forming ·NO2 radical (NO + cPTIO → ·NO2 + cPTI), which in turn can react with NO to form N2O3 (·NO2 + NO → N2O3). Thus, a reliable substitute NO quencher is required for various applications. Our recently published observations provided some evidence for the probable role of hemoglobin added in the growth medium on its ability to quench endogenous NO in sunflower seedlings [21, 22].

Ubiquitous occurrence of non-symbiotic hemoglobin (Hb) suggests that it serves important functions in the regulation of plant metabolism [23,24,25,26,27,28,29,30,31]. Endogenous hemoglobin primarily transports oxygen to various regions. Hbs reversibly bind with oxygen and their rates of binding and dissociation differ depending on the type of Hb. It also binds and scavenges NO and regulates its bioavailability in the tissues. In Arabidopsis thaliana, non-symbiotic class 1 and 2 Hbs reduce nitrite to NO, and this reaction rate increase linearly with [H+] increasing [32]. NO thus produced exhibits a strong affinity for the ferrous heme, leading to the formation of iron-nitrosyl-heme complex (Fe(II)-NO) as the final reaction product [33]. Hemoglobin scavenges NO through dioxygenation reaction where NO reacts with oxygenated hemoglobin (OxyHb; HbO2) to produce methemoglobin (MetHb; in which heme iron is in ferric state) and nitrate. This reaction occurs at the rate of 6–8 × 107 M−1 s−1 [34].

$${\text{HbFe(II)O}}_{ 2} + {\text{NO}} \to {\text{MetHb}} + {\text{NO}}_{3}^{ - }$$

Deoxygenated hemoglobin i.e. hemoglobin with ferrous heme iron, can also bind NO [35, 36]. Under these conditions, NO is no longer available for physiological functions in the tissues.

$${\text{HbFe(II)}} + {\text{NO}}\underset{\text{Slow}}{\overset{\text{Fast}}{\rightleftarrows}}{\text{HbFe(II)NO}}$$

These observations form the basis of current investigations to demonstrate the application of hemoglobin in the bathing medium as an effective scavenger of NO released from live plant tissue. The evidence from the present work demonstrates the ability of hemoglobin to scavenge NO from all cellular and apoplastic components of the tissue system. The methodology thus proposed offers an alternative approach to scavenge endogenous NO in various pharmacological studies in plants.

Materials and methods

Plant growth conditions

Sunflower seeds (Helianthus annuus L., var. KBSH 54) were washed with a liquid detergent (teepol) under running tap water, disinfected using 0.005% mercuric chloride and again washed under running tap water for 1 h. Seeds were then imbibed in distilled water for 2 h and placed on moist germination sheets irrigated with half-strength Hoagland nutrient solution. Seedlings were grown up to 2 days in dark at 25 °C. Sunflower seedlings showing uniform growth pattern were selected for various analyses.

Analysis of relative NO quenching ability of hemoglobin

Concentrated stock solution of hemoglobin (Sigma-aldrich, USA) was prepared fresh in distilled water for immediate use. To estimate the NO quenching ability of exogenously applied hemoglobin, 2 d old seedling roots were incubated for 30 min in dark in the absence or presence of variable concentrations of hemoglobin ranging from 250 μM to 3 mM. Each tube contained three seedlings. NO released from seedling root in the bathing solution was analyzed using MnIP-Cu (a copper derivative of 4-methoxy-2-(1H-naphtho (2,3-d) imidazol-2-yl) phenol; MNIP-Cu [37]. Seedling roots dipped only in distilled water were served as control. Following incubation, the bathing solution from each tube was taken for estimation of NO released in solution by treating with 2.5 µM of MnIP-Cu. NO released was monitored spectrofluorometrically (ex. 385 nm, em. 492 nm) and relative change in fluorescence was plotted to evaluate relative extent of NO released from tissue and quenched by variable concentrations of hemoglobin in solution.

Estimation of methemoglobin formation

Methemoglobin formation as a result of reaction between NO released from seedling roots and hemoglobin present in the solution was monitored spectrophotometrically at 406 nM. Oxygenated hemoglobin absorbs at a wavelength of 415 nM and its reaction with NO lead to the formation of methemoglobin which shifts the absorbance of the product (metHb) to 406 nM.

Visualization of NO in seedling roots in the absence or presence of hemoglobin

To further validate the scavenging ability of exogenously provided hemoglobin, seedling roots were dipped in distilled water containing variable concentrations of hemoglobin (2–3 mM) for 30 min in dark. Seedling roots dipped in distilled water served as control. After incubation, seedling roots were then incubated in 50 µM of MnIP-Cu for 45 min (ex. 385 nm, em. 492 nm). Using confocal laser scanning microscopy (CLSM; Leica, Germany), root tips were visualized for NO localization both in the absence or presence of hemoglobin.

Detection of nuclei in seedling roots using CLSM

Seedling roots were incubated with 4,6-diamidino-2-phenylindole (DAPI; 2 µg ml−1 in distilled water) for 2 min to localize nuclei using CLSM (ex. 360 nm and em. 460 nm).

Co-localization of NO and mitochondria in the seedling roots

Seedling roots were incubated in 50 µM of MnIP-Cu solution for 45 min and then dipped in 300 nM of MitoTracker (Molecular Probes, USA) for 45 min. NO and mitochondria signals were co-localized in the root-tip tissues by CLSM at ex. 385 nm; em. 492 nm for NO and at ex. 554 nm; em. 576 nm for mitochondria. Co-localization rate and mean intensity of co-localization of NO and mitochondrial signal were calculated using software LAS-AF, version 2.7-9723.3.

Statistical analysis

All experiments were performed at least thrice and statistically analyzed by SPSS 22.0 statistical program (SPSS Inc, Chicago, IL, U.S.A.) using One-Way ANOVA.


A novel fluorescence probe (a copper derivative of 4-methoxy-2-(1H-naphtho (2,3-d) imidazol-2-yl) phenol; MNIP-Cu; Fig. 1a) developed in the author’s laboratory in recent past for spectrofluorometric quantification and visualization of NO in live cells [37], has been used in the present work to examine the NO quenching ability of hemoglobin provided in the bathing medium. NO released from 2 d old, dark-grown sunflower seedling roots were monitored in the absence or presence of variable concentrations of hemoglobin (250 µM–3 mM) in the bathing solution. Since hemoglobin in solution binds with NO released from roots, resulting in methemoglobin (Hb-FeIII) formation and conversion of NO to NO3−, a Hb concentration-dependent decrease in the availability of free NO in solution is evident (Fig. 1b, c). Three millimoles of Hb leads to quenching of as much as 40% of NO released from roots as compared to control, thereby demonstrating the ability of externally available Hb to serve as a quencher of NO released from the tissue.

Fig. 1
figure 1

a Mechanism of action of MnIP-Cu, a novel probe for detection of NO in plant systems. b Quenching of NO released from seedling roots by exogenous hemoglobin (Hb) in a concentration (Hb)-dependent manner. NO released in solution from seedling roots bathed in varied concentrations of Hb was estimated using NO specific probe MnIP-Cu ex. 330 nm, em. 460 nm. c Conversion of oxyhemoglobin (HbO2) to methemoglobin in the presence of NO. d Absorbance peak for formation of methemoglobin in the presence of NO released from seedling roots in the solution bathed in varying concentrations of hemoglobin (250 µM to 3 mM) separately. e Increase in methemoglobin formation as a consequence of NO release from seedling roots in the bathing solution containing variable concentrations of hemoglobin (Hb). Note: Lower dosage of Hb in solution is insufficient in quenching NO from seedling roots

Exogenous Hb (λmax 415 nM) per se does not cross cell membranes (being a high molecular mass molecule of 64.5 kDa) but it can easily bind diffusible endogenous NO in a concentration (250 µM to 3 mM)-dependent manner and make it inaccessible as a free molecule in the bathing medium (forming methemoglobin; λmax 406 nM) (Fig. 1d, e). This observation on Hb as a NO quencher carries significance for pharmacological investigations in plant cells where, so far, cPTIO have been extensively used as NO quenchers. Figures 2 and 3 provide detailed evidence for quenching of endogenous NO from sunflower seedlings root cells in response to externally provide Hb (2–3 mM). In addition to cytoplasm and apoplast, NO has also been localized in nuclei and mitochondria. The ability of exogenous Hb to trigger migration of NO from all these intracellular locations thus proves its (Hbs) scavenging ability (for NO) from all intracellular locations of the plant cells/tissues exposed to various pharmacological investigations.

Fig. 2
figure 2

a Extracellular hemoglobin (Hb) as a quencher of endogenous NO from sunflower seedling roots. Seedling roots incubated in hemoglobin solution (2 mM and 3 mM) for 1 h followed by incubation in NO specific probe MnIP-Cu for 1 h. CLSM visualization of NO signal showed Hb concentration-dependent quenching of tissue NO. Roots were obtained from 2 day old dark-grown sunflower seedlings. Incubation medium without hemoglobin acts as control. b NO localization using CLSM in nuclei of root cells. Roots were obtained from 2 day old dark-grown sunflower seedlings. NO signal was visualized using MnIP-Cu. c Nuclei were visualized using DAPI. N Nuclei

Fig. 3
figure 3

Co-localization of NO and mitochondria in 2 day old, dark-grown sunflower seedling roots. Data analysis was done using software LAS-AF, version 2.7-9723.3. Co-localization rate—51.44%. Mean intensity of co-localization of NO signal—41.41. Mean intensity of co-localization of Mitochondrial signal—37.39


Hemoglobin is one of the hemoproteins, and NO is considered as a major regulatory component of the function of hemoproteins. NO can either activate or inhibit the activities of various hemoproteins by binding at the metallic center of heme. Furthermore, it is the oxidation state and the coordination environment of the iron center in the hemoproteins which determines the kinetics of NO binding with them [38]. Hexacoordination of heme molecule in non-symbiotic hemoglobin in plants enables it to bind with NO and scavenge it during hypoxic stress conditions [26]. Non-symbiotic hemoglobins possess ligand-binding characteristics different from that of symbiotic hemoglobins. Non-symbiotic Hbs exhibit high rate of oxygen binding than its rate of dissociation compared to symbiotic Hb, which possess high rate of oxygen binding as well as its dissociation. This difference in ligand-binding efficiency of the two proteins is due to differences in heme-coordination state. Thus, in non-symbiotic Hb, heme molecule is hexacoordianted compared to symbiotic Hb where it is pentacoordinated. Hemoglobin binds with both NO and O2 depending on the coordination state of heme molecule and performs the functions of either transportation of O2 or turnover/scavenging of NO. Symbiotic and erythrocyte hemoglobin is pentacoordinated which allows reversible binding of O2. Thus, they are capable of O2 transport and storage. However, non-symbiotic Hbs are hexacoordinated and exhibit very high avidity for O2. It exist as oxyhemoglobin under most physiological conditions and can efficiently scavenge NO via NO-dioxygenase activity [39,40,41,42,43]. Non-symbiotic hemoglobin expression is affected by a variety of stress conditions, such as hypoxia, cold stress and levels of cellular ATP [26, 27, 31]. In Arabidopsis, non-symbiotic Hb (AHb1) exhibits NOD activity in the presence of oxygen (Fig. 4). It removes NO using NADPH as electron donor leading to generation of nitrate and ferric hemoglobin, known as methemoglobin (metHb) [44]. High expression of nsHb in plants exhibits lot of significance as it enables plants to regulate high levels of NO, formed as a result of stress conditions, either by converting NO to nitrate via dioxygenation reaction or by forming nitrosylhemoglobin. Furthermore, it has been suggested that plant lines expressing high levels hemoglobin can prove to be better adapted to both normal and stress conditions [43].

Fig. 4
figure 4

Mechanism of non-symbiotic hemoglobin association with oxygen and NO in anoxic and oxygenic conditions. NOD NO-dioxygenase

Oxygenated hemoglobin (HbO2) is considered as a good choice, and can effectively scavenge NO within concentration range from 125 to 500 µM from both sunflower seedling roots and cotyledons tissue homogenates [21]. The reaction between NO and HbO2 is rapid, stoichiometric and leads to formation of methemoglobin and nitrate (NO3−) [45]. Due to its size (64.5 kDa), HbO2 does not cross cell membranes but can facilitate free diffusion of endogenous NO and its subsequent scavenging in the bathing medium (present work). Furthermore, HbO2 works well with the externally applied NO donor which generate NO even in the extracellular compartments, such as NONOates than those which needs intracellular bioactivation to release NO, like organic nitrates. Also, HbO2 does not inhibit transnitrosation reactions.


In view of the above-stated features of hemoglobin and current observations, it is a more reliable alternative to cPTIO as a NO scavenger of tissue NO in pharmacological investigations in plant systems. It (Hb) works efficiently in a concentration-dependent manner in efficiently quenching NO from plant tissues unlike cPTIO, which behaves differently (as NO quencher or as a source of NO) depending on its concentration in the medium. Thus, hemoglobin can be used as an efficient probe for estimation of NO emission from living tissues.



nitric oxide


2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide




copper derivative of 4-methoxy-2-(1H-naphthol (2,3-d) imidazol-2-yl) phenol


non-symbiotic hemoglobin




  1. Wildt J, Kley D, Rockel A, Rockel P, Segschneider HJ. Emission of NO from several higher plant species. J Geophys Res Atmos. 1997;102:5919–27.

    Article  CAS  Google Scholar 

  2. Chen J, Wu FH, Liu TW, Liu TW, Chen L, Xiao Q, Dong XJ, He JX, Pei ZM, Zheng HL. Emissions of nitric oxide from 79 plant species in response to simulated nitrogen deposition. Environ Pollut. 2012;160:192–200.

    Article  CAS  Google Scholar 

  3. Klepper LA. Nitric oxide (NO) and nitrogen dioxide (NO2) emissions from herbicide-treated soybean plants. Atmos Environ. 1979;13:537–42.

    Article  CAS  Google Scholar 

  4. Lea US, Ten Hoopen F, Provan F, Kaiser WM, Meyer C, Lillo C. Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in high nitrite excretion an NO emission from leaf and root tissue. Planta. 2004;219:59–65.

    Article  CAS  Google Scholar 

  5. Planchet E, Jagadis Gupta K, Sonoda M, Kaiser WM. Nitric oxide emission from tobacco leaves and cell suspensions: rate limiting factors and evidence for the involvement of mitochondrial electron transport. Plant J. 2005;41:732–43.

    Article  CAS  Google Scholar 

  6. Gupta KJ, Stoimenova M, Kaiser WM. In higher plants, only root mitochondria, but not leaf mitochondria reduce nitrite to NO, in vitro and in situ. J Exp Bot. 2005;56:2601–9.

    Article  CAS  Google Scholar 

  7. Chen J, Xiao Q, Wu FH, Pei ZM, Wang J, Wu YG, Zheng HL. Nitric oxide emission from barley seedlings and detached leaves and roots treated with nitrate and nitrite. Plant Soil Environ. 2010;56:201–8.

    Article  CAS  Google Scholar 

  8. Pfeiffer S, Mayer B, Hemmens B. Nitric oxide: chemical puzzles posed by a biological messenger. Angew Chem Int Ed Engl. 1999;38:1714–31.

    Article  Google Scholar 

  9. Neill SJ, Desikan R, Clarke A, Hancock JT. Nitric oxide signaling in plants. New Phytol. 2003;159:11–35.

    Article  CAS  Google Scholar 

  10. Lamattina L, Garcıa-Mata C, Graziano M, Pagnussat G. Nitric oxide: the versatility of an extensive signal molecule. Annu Rev Plant Biol. 2003;54:109–36.

    Article  CAS  Google Scholar 

  11. Shapiro AD. Nitric oxide signaling in plants. Vitam Horm. 2005;72:339–98.

    Article  CAS  Google Scholar 

  12. Corpas FJ, Barroso JB, Carreras A, Valderrama R, Palma JM, del Río LA. Nitrosative stress in plants: a new approach to understand the role of NO in abiotic stress. In: Lamattina L, Polacco JC, editors. Nitric oxide in plant growth, development and stress physiology. Plant cell monographs, vol. 5. Berlin: Springer; 2006.

    Google Scholar 

  13. Guo FQ, Okamoto M, Crawford NM. Identification of a plant nitric oxide synthase gene involved in hormonal signaling. Science. 2003;302:100–4.

    Article  CAS  Google Scholar 

  14. Bethke PC, Badger MR, Jones RL. Apoplastic synthesis of nitric oxide by plant tissues. Plant Cell. 2004;16:332–41.

    Article  CAS  Google Scholar 

  15. Corpas FJ, Barroso JB, del Río LA. Enzymatic sources of nitric oxide in plant cells: beyond one protein-one function. The New Phytologist. 2004;162:246–8.

    Article  CAS  Google Scholar 

  16. Guo FQ, Crawford NM. Arabidopsis nitric oxide synthase1 is targeted to mitochondria and protects against oxidative damage and dark-induced senescence. Plant Cell. 2005;17:3436–50.

    Article  CAS  Google Scholar 

  17. Gupta KJ, Kaiser WM. Production and scavenging of nitric oxide by Barley root mitochondria. Plant Cell Physiol. 2010;51:576–84.

    Article  CAS  Google Scholar 

  18. Gupta KJ, Fernie AR, Kaiser WM, van Dongen JT. On the origins of nitric oxide. Trends Plant Sci. 2011;16:160–8.

    Article  CAS  Google Scholar 

  19. Recalde L, Vázquez A, Groppa MD, Benavides MP. Reactive oxygen species and nitric oxide are involved in polyamine-induced growth inhibition in wheat plants. Protoplasma. 2018;255:1295.

    Article  CAS  Google Scholar 

  20. Arita NO, Cohen MF, Tokuda G, Yamasaki H. Fluorometric detection of nitric oxide with diaminofluoresceins (DAFs): applications and limitations for plant NO research. Plant Cell Monogr. 2006;5:269–80.

    Article  Google Scholar 

  21. Singh N, Bhatla SC. Signaling through reactive oxygen and nitrogen species is differentially modulated in sunflower seedling root and cotyledon in response to various nitric oxide donors and scavengers. Plant Signal Behav. 2017;12:e1365214.

    Article  Google Scholar 

  22. Singh N, Bhatla SC. Nitric oxide regulates lateral root formation through modulation of ACC oxidase activity in sunflower seedlings under salt stress. Plant Signal Behav. 2018;13:1–7.

    Google Scholar 

  23. Hebelstrup KH, Hunt P, Dennis E, Jensen SB, Jensen EØ. Hemoglobin is essential for normal growth of Arabidopsis organs. Physiol Plant. 2006;127:157–66.

    Article  CAS  Google Scholar 

  24. Bhattacharya S, Sen A, Thakur S, Tisa LS. Characterization of haemoglobin from Actinorhizal plants—an in silico approach. J Biosci. 2013;38:777–87.

    Article  CAS  Google Scholar 

  25. Dordas C, Rivoal J, Hill RD. Plant haemoglobins, nitric oxide and hypoxic stress. Ann Bot. 2003;91:173–8.

    Article  CAS  Google Scholar 

  26. Dordas C, Hasinoff BB, Igamberdiev AU, Manac’h N, Rivoal J, Hill RD. Expression of a stress-induced hemoglobin affects NO levels produced by alfalfa root cultures under hypoxic stress. Plant J. 2003;35:763–70.

    Article  CAS  Google Scholar 

  27. Dordas C, Hasinoff BB, Rivoal J, Hill RD. Class-1 haemoglobins, nitrate and NO levels in anoxic maize cell-suspension cultures. Planta. 2004;219:66–72.

    Article  CAS  Google Scholar 

  28. Vigeolas H, Huhn D, Geigenberger P. Nonsymbiotic haemoglobin-2 leads to an elevated energy state and to a combined increase in polyunsaturated fatty acids and total oil content when overexpressed in developing seeds of transgenic Arabidopsis plants. Plant Physiol. 2011;155:1435–44.

    Article  CAS  Google Scholar 

  29. Hebelstrup KH, Shah JK, Igamberdiev AU. The role of nitric oxide and haemoglobin in plant development and morphogenesis. Physiol Plant. 2013.

    Article  PubMed  Google Scholar 

  30. Kuruthukulangarakoola GT, Zhang J, Albert A, Winkler B, Lang H, Buegger F, Gaupels F, Heller W, Michalke B, Sarioglu H, Schnitzler JP, Hebelstrup KH, Durner J, Lindermayr C. Nitric oxide-fixation by non-symbiotic haemoglobin proteins in Arabidopsis thaliana under N-limited conditions. Plant Cell Environ. 2017;40:36–50.

    Article  CAS  Google Scholar 

  31. Vishwakarma A, Kumari A, Mur LAJ, Gupta K. A discrete role for alternative oxidase under hypoxia to increase nitric oxide and drive energy production. Free Radic Biol Med. 2018.

    Article  PubMed  Google Scholar 

  32. Tiso M, Tejero J, Basu S, Azarov I, Wang X, Simplaceanu V, Frizzell S, Jayaraman T, Geary L, Shapiro C, Ho C, Shiva S, Kim-Shapiro DB, Gladwin MT. Human neuroglobin functions as a redox-regulated nitrite reductase. J Biol Chem. 2011;286:18277–89.

    Article  CAS  Google Scholar 

  33. Tiso M, Tejero J, Kenney C, Frizzell S, Gladwin MT. Nitrite reductase activity of non-symbiotic hemoglobins from Arabidopsis thaliana. Biochemistry. 2012;51:5285–92.

    Article  CAS  Google Scholar 

  34. Kim-Shapiro DB, Schecter AN, Gladwin MT. Unrevealing the reactions of nitric oxide, nitrite, and hemoglobin in physiology and therapeutics. Arterioscler Thromb Vasc Biol. 2006;26:697–705.

    Article  CAS  Google Scholar 

  35. Cassoly R, Gibson QH. Conformation, co-operativity and ligand binding in human hemoglobin. J Mol Biol. 1975;91:301–13.

    Article  CAS  Google Scholar 

  36. Cooper CE. Nitric oxide and iron proteins. Biochim Biophys Acta. 1999;1411:290–309.

    Article  CAS  Google Scholar 

  37. David A, Yadav S, Baluška F, Bhatla SC. Nitric oxide accumulation and protein tyrosine nitration as a rapid and long distance signalling response to salt stress in sunflower seedlings. Nitric Oxide. 2015;50:28–37.

    Article  CAS  Google Scholar 

  38. Arora D, Jain P, Singh N, Kaur H, Bhatla SC. Mechanisms of nitric oxide crosstalk with reactive oxygen species scavenging enzymes during abiotic stress tolerance in plants. Free Radic Res. 2015;50:291–303.

    Article  Google Scholar 

  39. Hill RD. What are hemoglobins doing in plants? Can J Bot. 1998;76:707–12.

    CAS  Google Scholar 

  40. Igamberdiev AU, Hill RD. Nitrate, NO and haemoglobin in plant adaptation to hypoxia: an alternative to classic fermentation pathways. J Exp Bot. 2004;55:2473–82.

    Article  CAS  Google Scholar 

  41. Igamberdiev AU, Baron K, Manac’h-little N, Stoimenova M, Hill RD. The haemoglobin/nitric oxide cycle: involvement in flooding stress and effects on hormone signalling. Ann Bot. 2005;96:557–64.

    Article  CAS  Google Scholar 

  42. Gupta KJ, Hebelstrup KH, Mur LAJ, Igamberdiev AU. Plant hemoglobins: important players at the crossroads between oxygen and nitric oxide. FEBS Lett. 2011;585:3843–9.

    Article  CAS  Google Scholar 

  43. Riquelme A, Hinrichsen P. Non-symbiotic hemoglobin and its relation with hypoxic stress. Chil J Agric Res. 2015.

    Article  Google Scholar 

  44. Perazzolli M, Dominici P, Romero-Puertas MC, Zago E, Zeier J, Sonoda M, Lamb C, Delledonne M. Arabidopsis nonsymbiotic hemoglobin AHb1 modulates nitric oxide bioactivity. Plant Cell. 2004;16:2785–94.

    Article  CAS  Google Scholar 

  45. Doyle MP, Hoekstra JW. Oxidation of nitrogen-oxides by bound dioxygen in hemoproteins. J Inorg Biochem. 1981;14:351–8.

    Article  CAS  Google Scholar 

Download references

Authors’ contributions

Planning: Both the authors; Experimental work: NS; Writing of manuscript: both the authors. Both authors read and approved the final manuscript.


The authors are grateful to Joint UGC-Israel Science Foundation Research Project [F. No. 6-9/2017(IC)] for providing research funds.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Satish C. Bhatla.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Singh, N., Bhatla, S.C. Hemoglobin as a probe for estimation of nitric oxide emission from plant tissues. Plant Methods 15, 39 (2019).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: