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Nitric oxide (NO) produced in plant cells has the unique ability to interact with various other biomolecules, thereby facilitating its own as well as their signaling and associated actions at their sites of biosynthesis and at other sites via transcellular long distance transport of the molecular complexes. Melatonin (Mel) is one such biomolecule produced in plant cells which has fascinated plant biologists with regard to its molecular crosstalk with other molecules to serve its roles as a growth regulator. Present work reports the synthesis of N-nitrosomelatonin (NOMela) and its preferential uptake by Arabidopsis seedlings roots and long distance transport to the leaves through vascular strands. Equimolar (250 μM) concentrations of NOMela and S-nitrosoglutathione (GSNO) in aqueous solutions bring about 52.8% more release of NO from NOMela than from GSNO. Following confocal laser scanning microscopic (CLSM) imaging, Pearson's correlation coefficient analysis of the Scatter gram of endogenously taken up NOMela demonstrates significant NO signal in roots emanating from mitochondria. NOMela (250 μM) taken up by Arabidopsis seedling roots also proved more efficient as a NO transporter from primary root to leaves than 250 μM of GSNO. These novel observations on NOMela thus hold promise to decipher its crucial role as a NO carrier and reservoir in plant cells, and also as a facilitator of melatonin action in plant development., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Nitric oxide (NO) is an endogenous signaling molecule in plants. Sodium nitroprusside (SNP), an established NO donor used in plant science research, simultaneously releases NO, cyanide (CN - ) and iron (Fe) in solution. Since cyanide and iron mask NO effect of SNP, its use in NO research is debatable. Deciphering the action of SNP through NO, CN - or Fe has been undertaken in the present work. Cotyledons from salt stressed sunflower seedlings grown in the presence of NO donors were subjected to spectrofluorometric analysis of NO, CN - and Fe contents, and proteome and biochemical analyses. Diethylenetriamine NONOate (DETA) proved to be a better NO source since SNP enhanced ROS accumulation in the tissue. Abundance of 127 proteins is modulated by salt stress. SNP and exhausted SNP (exSNP) alter the abundance of 117 and 129 proteins, respectively. These proteins belong to primary metabolism, stress-response, transport, translation, proteolysis, chaperone, regulatory, and storage. Salt-responsive proteins, such as, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK) and isocitrate dehydrogenase are negatively modulated. DETA and SNP lower the activities of GAPDH and S-adenosylmethionine synthase (SAMS). Abundance of heat shock 70 kDa protein and actin are sensitive to both NaCl and NO. SNP affects plant growth by modulating proteome though iron, cyanide and NO. Its use only as an NO donor is thus debatable. exSNP control also releases substantial amount of cyanide and iron, thus questioning its use as control in NO research., (Copyright © 2019. Published by Elsevier Inc.)

The present findings demonstrate significant modulation of total glutathione content, reduced glutathione (GSH) content, oxidized glutathione (GSSG) content, GSH/GSSG ratio and glutathione reductase (GR; EC 1.6.4.2) activity in dark-grown seedling cotyledons in response to salt-stress (120 mM NaCl) in sunflower (Helianthus annuus L.) seedlings. A differential spatial distribution of GR activity (monitored by confocal laser scanning microscopic (CLSM) imaging) is also evident. Melatonin and nitric oxide (NO) differentially ameliorate salt stress effect by modulating GR activity and GSH content in seedling cotyledons. Total glutathione content (GSH + GSSG) exhibit a seedling age-dependent increase in the cotyledons, more so in salt-stressed conditions and when subjected to melatonin treatment. Seedlings raised in presence of 15 μM of melatonin exhibit significant increase in GR activity in cotyledon homogenates (10,000 g supernatant) coinciding with significant increase in GSH content. GSSG content and GSH/GSSG ratio also increased due to melatonin treatment. A correlation is thus evident in NaCl-sensitized modulation of GSH content and GR activity by melatonin. GSH content is down regulated by NO provided as 250 μM of sodium nitroprusside (SNP) although total glutathione content remained in similar range. A reversal of response (enhanced total glutathione accumulation) by NO scavenger (cPTIO) highlights the critical role of NO in modulating glutathione homeostasis. SNP lowers the activity of hydroxyindole-O-methyltransferase (HIOMT) - a regulatory enzyme in melatonin biosynthesis in control seedlings whereas its activity is upregulated in salt-stressed seedling cotyledons. Melatonin content of seedling cotyledons is also modulated by NO. NO and melatonin thus seem to modulate GR activity and GSH content during seedling growth under salt stress., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Nitric oxide is a significant component of iron signaling in plants. Heme is one of the iron sensors in plants. Free heme is highly toxic and can cause cell damage as it catalyzes the formation of reactive oxygen species (ROS). Its catabolism is carried out by heme oxygenase (HOs; EC 1.14.99.3) which uses heme both as a prosthetic group and as a substrate. Two significant events, which accompany adaptation to salt stress in sunflower seedlings, are accumulation of ROS and enhanced production of nitric oxide (NO) in roots and cotyledons. Present investigations on the immunolocalization of heme oxygenase distribution in sunflower seedling cotyledons by confocal laser scanning microscopic (CLSM) imaging provide new information on the differential spatial distribution of the inducible form of HO (HO-1) as a long distance in response to NaCl stress. The enzyme is abundantly distributed in the specialized cells around the secretory canals (SCs) in seedling cotyledons. Abundance of tyrosine nitrated proteins has also been observed in the specialized cells around the secretory canals in cotyledons derived from salt stressed seedlings. The spatial distribution of tyrosine nitrated proteins and HO-1 expression further correlates with the abundance of mitochondria in these cells. Present findings, thus, highlight a link among distribution of HO-1 expression, abundance of tyrosine nitrated proteins and mitochondria in specialized cells around the secretory canal as a long distance mechanism of salt stress tolerance in sunflower seedlings. Enhanced spatial distribution of HO-1 in response to NaCl stress in seedling cotyledons is in congruence with the observed increase in specific activity of HO-1 in NaCl stressed conditions. The enzyme activity is further enhanced by hemin (HO-1 inducer) both in the absence or presence of NaCl stress and inhibited by zinc protoporphyrin. Western blot analysis of cotyledon homogenates using anti-HO-1 polyclonal antibody shows one major band (29 kDa) of HO-1. NaCl-modulated HO-1 activity correlates with endogenous NO content in the cotyledons. Increased NO accumulation by hemin treatment also correlates with enhanced activity of HO-1 in both control and NaCl stress conditions. Present work indicates that NO positively modulates HO-1 activity in sunflower seedling cotyledons. NaCl stress tends to antagonize NO action on HO-1 activity. NO (from sodium nitroprusside; SNP) is probably positively modulating HO-1 activity by way of its interaction/binding with heme group. Present work also shows enhanced NO accumulation in seedling cotyledons both in the absence or presence of iron in the growth medium, in response to NaCl stress. Thus, a probable link between endogenous NO, NaCl stress and iron-homeostasis by way of modulation of HO-1 activity at early stage of sunflower seedling growth has been proposed., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Sensing of salt stress by sunflower seedlings accompanies temporal and spatial modulation of intracellular nitric oxide (NO) accumulation and protein tyrosine nitration as markers of nitrosative stress. Employing a novel NO-specific probe for NO localization (a copper derivative of 4-methoxy-2-(1H-naphtho(2,3-d)imidazol-2-yl)phenol; MNIP-Cu) synthesized in author's laboratory, immunological analysis of tyrosine nitrated proteins by confocal laser scanning microscopy (CLSM) and Western blot analysis, these rapid signalling events have been investigated. MNIP-Cu reveals the distribution of NO in whole seedlings. Preferential and enhanced NO localization around oil bodies (OBs) in cotyledons within 48 h of salt-stressed seedlings exhibits rapid transport of nitrosative stress signal from roots to the cotyledons. Immunological analysis reveals enhanced gradient of tyrosine nitrated proteins in salt-stressed roots from tip to the differentiating zone and from columella to the deep-seated cells. Western blot analysis shows that at least eight major cytosolic proteins exhibit enhanced tyrosine nitration in seedling roots in response to salt stress. Present observations provide strong evidence for rapid NO accumulation in salt stressed sunflower seedling roots and cotyledons and its impact on enhanced tyrosine nitration of cytosolic and OB proteins, as a mechanism to provide longevity to OBs for seedling survival under the salt stress., (Copyright © 2015 Elsevier Inc. All rights reserved.)