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Urban air pollution has been a global challenge world-wide. While urban vegetation or forest modelling can be useful in reducing the toxicities of the atmospheric gases by their absorption, the surge in gaseous pollutants negatively affects plant growth, thereby altering photosynthetic efficiency and harvest index. The present review analyses our current understanding of the toxic and beneficial effects of atmospheric nitrogen oxides (NO x ), hydrogen sulphide (H 2 S) and carbon monoxide (CO) on plant growth and metabolism. The atmospheric levels of these gases vary considerably due to urbanization, automobile emission, volcanic eruptions, agricultural practices and other anthropological activities. These gaseous pollutants prevalent in the atmosphere are known for their dual action (toxic or beneficiary) on plant growth, development and metabolism. NO seems to exert a specialized impact by upregulating nitrogen metabolism and reducing tropospheric ozone. High H 2 S emission in specific areas of geothermal plants, fumarolic soils and wetlands can be a limitation to air quality control. Certain shortcomings associated with the designing of field experiments, sensitivity of detection methods and simulation development are yet to be overcome to analyze the precise levels of NO, H 2 S and CO in the rhizosphere of diverse agro-climatic regions. Several laboratory-based investigations have been undertaken to assess the roles of atmospheric gases, namely NO x , CO, H 2 S, and particulate matter (PM). However, in order to enable natural and sustainable mitigation, it is essential to increase the number of field experiments in order to identify the pollutant-tolerant plants and study their interactive impact on plant growth and agriculture., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

Newly discovered plant behaviors, linked to historical observations, contemporary technologies, and emerging knowledge of signaling mechanisms, argue that plants utilize complex information processing systems. Plants are goal-oriented in an evolutionary and physiological sense; they demonstrate agency and learning. While most studies on plant plasticity, learning, and memory deal with the responsiveness of individual plants to resource availability and biotic stresses, adaptive information is often perceived from and coordinated with neighboring plants, while competition occurs for limited resources. Based on existing knowledge, technologies, and sustainability principles, climate-smart agricultural practices are now being adopted to enhance crop resilience and productivity. A deeper understanding of the dynamics of plant behavior offers a rich palette of potential amelioration strategies for improving the productivity and health of natural and agricultural ecosystems.

Oil body (OB) mobilization, a crucial event associated with early seedling growth, is delayed in response to salt stress. Previous reports suggest that careful regulation of polyamine (PA) metabolism is essential for salt stress tolerance in plants. Many aspects of PA-mediated regulation of metabolism have been uncovered. However, their role in the process of OB mobilization remains unexplored. Interestingly, the present investigations reveal a possible influence of PA homeostasis on OB mobilization, while implicating complex regulation of oleosin degradation and aquaporin abundance in OB membranes in the process. Application of PA inhibitors resulted in the accumulation of smaller OBs when compared to control (-NaCl) and the salt-stressed counterparts, suggesting a faster rate of mobilization. PA deficit also resulted in reduced retention of some larger oleosins under controlled conditions but enhanced retention of all oleosins under salt stress. Additionally, with respect to aquaporins, a higher abundance of PIP2 under PA deficit both under control and saline conditions, is correlated with a faster mobilization of OBs. Contrarily, TIP1s, and TIP2s remained almost undetectable in response to PA depletion and were differentially regulated by salt stress. The present work, thus, provides novel insights into PA homeostasis-mediated regulation of OB mobilization, oleosin degradation, and aquaporin abundance on OB membranes.

Sustainable agriculture faces major challenges under abiotic stress conditions owing to extensive application of chemical fertilizers which pollute water, soil and atmosphere. Biostimulants (BSs), comprising of naturally derived complex mixtures of uncharacterized biomolecules, pure biochemicals and nanomaterials, enhance nutrient use efficiency (NUE) and trigger crop's natural defense mechanisms. While it is difficult to specify the metabolic effects of uncharacterized natural mixtures (seaweed extract, protein hydrolyzates, etc.), exogenous application of pure biochemicals and nanomaterials offers an edge as BSs since their physiological roles and mechanisms of action are decipherable. Foliar application or seed treatment of some amino acids, polyamines and biopolymers (chitosan, lipochitin oligosaccharides and thuricin 17) enable plants to overcome drought and salinity stress via activation of mechanisms for reactive oxygen species (ROS) scavenging, osmolyte regulation and chlorophyll accumulation. Interaction of nitric oxide (NO) with some vitamins and melatonin exhibits potential significance as BSs for mitigating stress by ROS scavenging and maintenance of intracellular ionic balance and membrane integrity. Near future is likely to see wide applications of nanoparticles (NPs) and nanomaterials (NMs) as BSs in view of their biphasic mode of action (bio-physical activation of membrane receptors followed by gradual release of BS into the plant cells).

Plant growth under abiotic stress conditions significantly enhances intracellular generation of reactive oxygen species (ROS). Oxidative status of plant cells is directly affected by the modulation of iron homeostasis. Among mammals and plants, heme oxygenase-1 (HO-1) is a well-known antioxidant enzyme. It catalyzes oxygenation of heme, thereby producing Fe 2+ , CO and biliverdin as byproducts. The antioxidant potential of HO-1 is primarily due to its catalytic reaction byproducts. Biliverdin and bilirubin possess conjugated π-electrons which escalate the ability of these biomolecules to scavenge free radicals. CO also enhances the ROS scavenging ability of plants cells by upregulating catalase and peroxidase activity. Enhanced expression of HO-1 in plants under oxidative stress accompanies sequestration of iron in specialized iron storage proteins localized in plastids and mitochondria, namely ferritin for Fe 3+ storage and frataxin for storage of Fe-S clusters, respectively. Nitric oxide (NO) crosstalks with HO-1 at multiple levels, more so in plants under oxidative stress, in order to maintain intracellular iron status. Formation of dinitrosyl-iron complexes (DNICs) significantly prevents Fenton reaction during oxidative stress. DNICs also release NO upon dissociation in target cells over long distance in plants. They also function as antioxidants against superoxide anions and lipidic free radicals. A number of NO-modulated transcription factors also facilitate iron homeostasis in plant cells. Plants facing oxidative stress exhibit modulation of lateral root formation by HO-1 through NO and auxin-dependent pathways. The present review provides an in-depth analysis of the structure-function relationship of HO-1 in plants and mammals, correlating them with their adaptive mechanisms of survival under stress., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Sunflower ( Helianthus annuus L.) is one of the major oilseed crops cultivated world over for its high-quality oil rich in linoleic acid. It also has established applications in pharmaceutical and biotechnological industries, mainly through recombinant production of unique oil body (OB) membrane proteins-oleosins, which are used for producing a wide variety of vaccines, food products, cosmetics and nutraceuticals. The present review provides a critical analysis of the progress made in advancing our knowledge in sunflower biology, ranging from mechanisms of pollen-stigma interaction, seed development, physiology of seed germination and seedling growth under salt stress, and finally understanding the signaling routes associated with various biochemical pathways regulating seedling growth. Role of nitric oxide (NO) triggered post-translational modifications (PTMs), discovered in the recent past, have paved way for future research directions leading to further understanding of sunflower developmental physiology. Novel protocols recently developed to monitor temporal and spatial distributions of various biochemicals involved in above-stated developmental events in sunflower, will go a long way for similar applications in plant biology in future.

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.)

Among abiotic stresses, salt stress is a major threat to crop production all over the world. Present work demonstrates the profuse accumulation of Na+ in 2-day-old, dark-grown sunflower (Helianthus annuus L.) seedlings roots in response to salt stress (NaCl). The pattern of K+ accumulation in response to salt stress is similar to that of Na+ but on relatively lower scale. Application of nitric oxide (NO) donor (DETA) scales down Na+ accumulation in salt-stressed seedlings. The impact of NO donor on K+ accumulation is, however, different in control and salt-stressed seedling roots. In control seedlings, it enhances K+ accumulation, whereas, it gets reduced in salt-stressed seedlings. Specialised channels called 'aquaporins' (AQPs) play a major role maintaining the water status and transport across plant parts under salt-stress. Thus, accumulation of plasma-membrane intrinsic proteins (PIPs) and tonoplast-intrinsic proteins (TIPs), localised on plasma-membrane and vacuolar-membrane, respectively was undertaken in 2-day-old, dark-grown seedling roots. Salt stress increased the abundance of these isoforms, whereas, NO application resulted in decreased accumulation of PIP2 and TIP1. PIP1 and TIP2 isoforms remained undetectable. Present work thus, puts forward a correlation between AQP expression and ions (Na+ and K+) homeostasis in response to salt stress and NO.

Salt stress adversely affects plants by causing osmotic and ionic imbalance. Cellular osmotic adjustment occurs by modulation of water fluxes. Polyamines (PAs) are often advocated to be involved in osmoregulation during stressful conditions, and thus, they serve as potential "osmolytes." Aquaporins (AQPs), the water-transporting channels, are expected to play crucial roles in osmoregulation. Present investigations on etiolated sunflower seedlings demonstrate a possible correlation between PA homeostasis and maintenance of water balance, as a function of modulation of the abundance of two major AQP subfamilies: PIP2 (plasma membrane intrinsic protein 2) and TIP1 (tonoplast intrinsic protein 1). Salt stress (120 mM NaCl) restricts growth of sunflower seedlings and induces reduction in relative water content (RWC). This accompanies enhanced abundance of PIP2s and TIP1s in seedling roots and that of TIP1s in cotyledons, as revealed by Western blot analysis of AQP isoforms and also their imaging by confocal laser scanning microscopy (CLSM). Raising seedlings in the presence of 500 μM of DFMA (DL-α-difluoromethylarginine) or DFMO (DL-α-difluoromethylornithine), which are potent inhibitors of PA biosynthesis enzymes (arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), respectively), significantly promotes root extension, irrespective of NaCl stress, and results in further lowering of salt-induced reduction in RWC in roots and cotyledons. This correlates with enhanced accumulation of both PIP2s and TIP1s in seedling roots, but not in cotyledons. Present work, therefore, implicates PA homeostasis in the maintenance of water status of sunflower seedlings, possibly via regulation of abundance and distribution of AQP isoforms associated with the plasma membrane and tonoplast.

Helianthus annuus L. is an important oilseed crop, which exhibits moderate salt tolerance and can be cultivated in areas affected by salinity. Using patch-clamp electrophysiology, we have characterized Na + influx and K + efflux conductances in protoplasts of salt-tolerant H. annuus L. hybrid KBSH-53 under high salinity. This work demonstrates that the plasma membrane of sunflower root cells has a classic set of ionic conductances dominated by K + outwardly rectifying channels (KORs) and non-selective cation channels (NSCCs). KORs in sunflower show extreme Na + sensitivity at high extracellular [Ca 2+ ] that can potentially have a positive adaptive effect under salt stress (decreasing K + loss). Na + influx currents in sunflower roots demonstrate voltage-independent activation, lack time-dependent component, and are sensitive to Gd 3+ . Sunflower Na + -permeable NSCCs mediate a much weaker Na + influx currents on the background of physiological levels of Ca 2+ as compared to other species. This suggests that sunflower NSCCs have greater Ca 2+ sensitivity. The responses of Na + influx to Ca 2+ correlates well with protection of sunflower growth by external Ca 2+ in seedlings treated with NaCl. It can be, thus, hypothesized that NaCl tolerance in sunflower seedling roots is programmed at the ion channel level via their sensitivity to Ca 2+ and Na + ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hryvusevich, Navaselsky, Talkachova, Straltsova, Keisham, Viatoshkin, Samokhina, Smolich, Sokolik, Huang, Yu, Bhatla and Demidchik.)

Present findings hypothesize that salt-tolerant and -sensitive oilseed plants are expected to exhibit deviant patterns of growth through lipolytic events in seedling cotyledons. It reports the growth response and different lipolytic mechanisms operating during oil body (OB) mobilization in the seedling cotyledons of salt-tolerant (DRSH 1) and salt-sensitive (PSH 1962) varieties of sunflower ( Helianthus annuus L.). Salt tolerance or sensitivity to 120 mM NaCl correlates with high proteolytic degradation of OB membrane proteins, particularly oleosins, whereas salt-sensitive seedling cotyledons exhibit negligible proteolytic activity, thereby retaining OB membrane integrity for a longer time. High lipoxygenase (LOX) activity and its further upregulation by salt stress are the unique features of salt-sensitive sunflower seedlings. Salt-tolerant seedling cotyledons exhibit noteworthy modulation of phospholipase-D (PLD) activity by salt stress. Salt-sensitive seedling cotyledons exhibit higher lipase activity than salt-sensitive ones and enzyme activity is downregulated by salt stress. Salt-sensitive variety exhibits higher lipid accumulation and faster lipid mobilization with seedling development than salt-tolerant variety. Accumulation of oleic and linoleic acid in the seedling cotyledons of salt-tolerant and sensitive varieties exhibits differential sensitivity to salt stress. Novel detection of hexanoic acid (6:0) is a noteworthy feature as a response to salt stress in salt-sensitive variety. These findings, thus, provide new information on long-distance salt stress sensing mechanisms at seedling stage of plant development.

Human beings and plants experience a variety of stress conditions and adapt themselves through novel molecular crosstalk in their cellular constituents. Nitric oxide (NO), haemoglobin and melatonin interact with each other not only in blood stream of human beings, but also in the cells and metabolically active conducting strands of plants. Specialised sites of biosynthesis and differential intracellular spatial distribution of these molecules have been clearly demonstrated by the authors in plant systems. This has led to an understanding of the role of these molecules under salt stress conditions experienced by plants: NO is a modulator of enzyme activity through S-nitrosylation and tyrosine nitration, haemoglobin (phytoglobin) is an NO scavenger, and melatonin is a reactive oxygen species (ROS) scavenger involved in key crosstalk in both plants and humans facing stress. Our recent work on heme oxygenase (HO) activity modulation by stress in plants, and its interaction with NO, further demonstrates common features of molecular crosstalk in protecting plants and human beings from stress.

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.)

Background: 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., Result: 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., Conclusion: 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., Competing Interests: The authors declare that they have no competing interests.

Glycine betaine (GB), an osmolyte, is produced in chloroplasts by the action of betaine aldehyde dehydrogenase (BADH) on its precursor betaine aldehyde. The present work highlights the significance of nitric oxide (NO) in GB homeostasis as a long-distance salt (120 mM NaCl) stress-elicited response. In light-grown seedling cotyledons, both the activity and transcript levels of BADH are much higher than in dark-grown seedlings irrespective of salt stress. Significantly high accumulation of GB in dark-grown seedling cotyledons indicates its preferential mobilization from cotyledons to other plant parts in light-grown seedlings. NO donor application (diethylenetriamine) maintains high BADH activity in light, although in dark it is brought down marginally. BADH levels are maintained high in light than in dark in respective treatments. Reversal of the effect of NO donor on age-dependent GB content, BADH activity, and transcript levels by NO scavenger (diethyldithiocarbamate) further demonstrates the impact of NO on GB homeostasis in light- and dark-grown seedlings in an age-dependent manner, major modulation being observed in 4-d-old seedlings. The present work, thus, provides new information on co-regulation of GB homeostasis by NO and light. It also puts forward new information of GB-NO crosstalk in maneuvering salt stress sensing as a long-distance response in seedlings.

Sunflower plants are semi-tolerant to salt stress. Calcium modulates the expression of oubain-sensitive ATPases, responsible for sodium fluxes in cells. Salt stress delays degradation of oil body (OB) membrane proteins. Serotonin and melatonin contents are elevated in response to salt stress. Melatonin can detoxify the seedlings of elevated reactive oxygen species (ROS) levels. Enhanced nitric oxide (NO) expression correlates with NaCl-induced modulation of seedling growth. Salt stress enhances S-nitrosylation of cytosolic proteins in seedling cotyledons, while in roots, denitrosylation of proteins is observed. Lipid peroxide content and glutathione peroxidase (GPX4) activity are enhanced in response to salt stress. Salt stress downregulates the activity of superoxide dismutase (SOD) and upregulates the activity of GPX4 and glutathione reductase (GR). Heme oxygenase-1 (HO-1) abundance in cells surrounding the secretory canal in seedling cotyledons is enhanced in response to salt stress. NO negatively regulates the total glutathione homeostasis and regulates polyamine and glycine betaine homeostasis in response to salt stress. An intricate biochemical crosstalk is thus observed to control salt tolerance mechanisms in sunflower.

Free polyamine (PA) titers in plants may be regulated through reversible conjugate formation and/or through modulation of their synthesis, transport and degradation. PA signaling involves the well-acknowledged signaling molecule, nitric oxide (NO), which functions in diverse biological processes. Present investigations demonstrate the influence of salt stress (120 mM NaCl) and exogenous NO donor (250 µM Diethylenetriamine, DETA) on PA homeostasis of 2 d old, etiolated sunflower ( Helianthus annuus L.) seedling cotyledons as a long-distance signaling response. Significantly enhanced intracellular spermine (Spm) accumulation was observed in seedling cotyledons under salt stress and in response to NO donor, the increase being more pronounced in seedlings treated with NO, evidently as a result of upregulation of the PA biosynthetic enzymes - arginine decarboxylase (ADC) and S -adenosylmethionine decarboxylase (SAMDC) - as revealed by Western blot and confocal imaging (CLSM). Moreover, salt stress induced the activity of polyamine oxidase (PAO), a PA catabolic enzyme, while NO lowered its activity in salt-stressed seedling cotyledons. NO, thus, appears to assist the seedlings in adapting to salt stress by positively regulating PA homeostasis through regulation of PA distribution between free, conjugated and bound forms, increased accumulation of PA biosynthetic enzymes and lowering the rate of PA catabolism.

Present work focuses on tissue and concentration-dependent effect of nitric oxide (NO) on the modulation of cytosolic peroxidase (POD; EC 1.11.1.7) activity in 2-day old etiolated sunflower (Helianthus annuus L.) seedlings. Exogenously supplied NO (in the form of sodium nitroprusside [SNP] or diethylenetriamine NONOate [DETA]; 125 to 500 μM) results in noteworthy enhancement in seedling growth in a concentration dependent manner irrespective of salt-stress and differentially affects POD activity in 2-day old seedling cotyledons. Elevated NO availability leads to an increase in the specific activity of POD in a concentration-dependent manner within 48 hrs as a rapid signaling response. Purification of POD protein using immunoprecipitation technique has shown that cotyledons derived from salt stressed seedlings exhibit a higher extent of tyrosine nitration of POD as compared to the control seedlings. Out of the four tyrosine residues found in the amino acid sequence of POD, the one at position 100 has been predicted to undergo nitration. Thus, a probable NO-POD crosstalk is evident in sunflower seedling cotyledons accompanying salt stress.

Understanding the mechanisms of sodium (Na⁺) influx, effective compartmentalization, and efflux in higher plants is crucial to manipulate Na⁺ accumulation and assure the maintenance of low Na⁺ concentration in the cytosol and, hence, plant tolerance to salt stress. Na⁺ influx across the plasma membrane in the roots occur mainly via nonselective cation channels (NSCCs). Na⁺ is compartmentalized into vacuoles by Na⁺/H⁺ exchangers (NHXs). Na⁺ efflux from the plant roots is mediated by the activity of Na⁺/H⁺ antiporters catalyzed by the salt overly sensitive 1 (SOS1) protein. In animals, ouabain (OU)-sensitive Na⁺, K⁺-ATPase (a P-type ATPase) mediates sodium efflux. The evolution of P-type ATPases in higher plants does not exclude the possibility of sodium efflux mechanisms similar to the Na⁺, K⁺-ATPase-dependent mechanisms characteristic of animal cells. Using novel fluorescence imaging and spectrofluorometric methodologies, an OU-sensitive sodium efflux system has recently been reported to be physiologically active in roots. This review summarizes and analyzes the current knowledge on Na⁺ influx, compartmentalization, and efflux in higher plants in response to salt stress., Competing Interests: The authors declare no conflict of interest.

Nitric oxide (NO) is established as a modulator of various developmental processes in plants through its interaction with multiple enzymatic and non-enzymatic biomolecules. Lateral root (LR) induction and extension in sunflower (Helianthus annuus L.) has been observed to be governed by a probable crosstalk between NO and ethylene biosynthesizing enzyme-ACC oxidase. NaCl (120 mM) stress not only lowers LR induction but also reduces their extension growth. Quenching of endogenous NO by raising seedlings in presence of 40 µM hemoglobin in the growth medium does not affect LR induction but lowers their extension growth. NaCl stress and NO depletion have additive effects on the enhancement of ACC oxidase activity, leading to enhanced ethylene biosynthesis. Role of NO has been further confirmed by raising sunflower seedlings in the presence of 20-60 µM of two NO donors, sodium nitroprusside (SNP) and diethylenetriamine NONOate (DETA). LR extension growth was higher with DETA than SNP as NO donor at 40 µM. Iron-deficiency also promoted LR proliferation. It also significantly lowered ACC oxidase activity in the seedling roots in response to salt stress. Based on the present findings it is proposed that salt stress-mediated LR proliferation is regulated by NO through its binding with ACC oxidase (an iron-containing enzyme). This results in the formation of a stable ternary complex (ACC-ACC oxidase-NO) which leads to the reduction in ethylene biosynthesis. Lesser availability of ethylene consequently brings about enhanced LR formation.

Tyrosine nitrated proteins can be detected in plant cells electrophoretically and their distribution can be monitored by confocal laser scanning microscopy (CLSM) imaging. One-dimensional polyacrylamide gel electrophoresis (1D PAGE) followed by Western blotting using polyclonal antibody against 3-nitrotyrosine residues enables detection of tyrosine nitrated proteins in plant cells. Here we describe detection of tyrosine nitrated proteins in the homogenates derived from sunflower (Helianthus annuus L.) seedling cotyledons. Total soluble proteins obtained from tissue homogenates are resolved using vertical gel electrophoresis followed by their electrophoretic transfer on to a microporous membrane support for immunodetection. Spatial distribution of tyrosine nitrated proteins can be visualized using an antibody against 3-nitrotyrosine residues. Immunofluorescent localization is performed by cutting 7 μm thick wax sections of tissue followed by incubation in primary anti-nitrotyrosine antibody (dilution 1:200) and secondary Cy-3 labeled anti-rabbit IgG antibody (dilution 1:1500). Confocal laser scanning microscopy analysis is undertaken using argon lasers (ex: 530-550 nm and em: 570 nm) at pinhole 1. Modulation in the abundance and spatial localization of tyrosine nitrated proteins in plant tissues can be monitored using these techniques.

Nitric oxide (NO) and various reactive nitrogen species produced in cells in normal growth conditions, and their enhanced production under stress conditions are responsible for a variety of biochemical aberrations. The present findings demonstrate that sunflower seedling roots exhibit high sensitivity to salt stress in terms of nitrite accumulation. A significant reduction in S-nitrosoglutathione reductase (GSNOR) activity is evident in response to salt stress. Restoration of GSNOR activity with dithioerythritol shows that the enzyme is reversibly inhibited under conditions of 120 mM NaCl. Salt stress-mediated S-nitrosylation of cytosolic proteins was analyzed in roots and cotyledons using biotin-switch assay. LC-MS/MS analysis revealed opposite patterns of S-nitrosylation in seedling cotyledons and roots. Salt stress enhances S-nitrosylation of proteins in cotyledons, whereas roots exhibit denitrosylation of proteins. Highest number of proteins having undergone S-nitrosylation belonged to the category of carbohydrate metabolism followed by other metabolic proteins. Of the total 61 proteins observed to be regulated by S-nitrosylation, 17 are unique to cotyledons, 4 are unique to roots whereas 40 are common to both. Eighteen S-nitrosylated proteins are being reported for the first time in plant systems, including pectinesterase, phospholipase d-alpha and calmodulin. Further physiological analysis of glyceraldehyde-3-phosphate dehydrogenase and monodehydroascorbate reductase showed that salt stress leads to a reversible inhibition of both these enzymes in cotyledons. However, seedling roots exhibit enhanced enzyme activity under salinity stress. These observations implicate the role of S-nitrosylation and denitrosylation in NO signaling thereby regulating various enzyme activities under salinity stress in sunflower seedlings., (© 2017 Scandinavian Plant Physiology Society.)

Nitric oxide (NO) signalling in plants is responsible for modulation of a variety of plant developmental processes. Depending on the tissue system, the signalling of NO-modulated biochemical responses majorly involves the processes of tyrosine nitration or S-nitrosylation of specific proteins/enzymes. It has further been observed that there is a significant impact of various biotic/abiotic stress conditions on the extent of tyrosine nitration and S-nitrosylation of various metabolic enzymes, which may act as a positive or negative modulator of the specific routes associated with adaptive mechanisms employed by plants under the said stress conditions. In addition to recent findings on the modulation of enzymes of primary metabolism by NO through these two biochemical mechanisms, a major mechanism for regulating the levels of reactive oxygen species (ROS) under stress conditions has also been found to be through tyrosine nitration or S-nitrosylation of ROS-scavenging enzymes. Recent investigations have further highlighted the differential manner in which the ROS-scavenging enzymes may be S-nitrosylated and tyrosine nitrated, with reference to their tissue distribution. Keeping in mind the very recent findings on these aspects, the present review has been prepared to provide an analytical view on the significance of protein tyrosine nitration and S-nitrosylation in plant development.

Sodium nitroprusside (SNP), diethylenetriamine NONOate (DETA), S-nitroso-n-acetyl-D,L- penicillamine (SNAP), and 4-(p-methoxyphenyl)-1,3,2- Oxathiazolylium-5-olate (CAY) exhibit differential NO releasing ability in aqueous solution and hemoglobin is a more efficient NO quencher than cPTIO in solution. DETA releases 16% more NO compared with SNP in solution. Various NO donors (SNP, DETA, SNAP, and CAY) also bring about a differential but concentration-dependent increase in endogenous NO in seedling cotyledons and roots. Two-day old, dark-grown seedling roots exhibit 95%, 77%, 59% and 45% increase in NO content in presence of each of 500 µM of DETA, SNAP, CAY and SNP, respectively, relative to control. NO accumulation in the tissue system as a response to NO donors is reflected in terms of corresponding peroxynitrite accumulation. Release of cyanide and free iron as byproducts of SNP dissociation in solution limits its usefulness as an NO donor. SNP leads to profuse ROS generation in sunflower seedling roots. Light is not a pre-requisite for NO generation from SNP. Present work also demonstrates the usefulness of hemoglobin over cPTIO as NO scavenger. Hemoglobin brings about increasing NO quenching with its increasing concentration from 2.5 to 10 µM. Greater sensitivity of the root system to the NO donor/scavenger treatments is evident, it being in direct contact with the molecules in the incubation/ growth medium. This differential effect does not seem to be significantly transmitted to the cotyledons (long-distance signaling).

Salinity results in significant reduction in sunflower (Helianthus annuus L.) seedling growth and excessive generation of reactive oxygen species (ROS). Present work highlights the possible role of melatonin as an antioxidant through its interaction with nitric oxide (NO), and as an early and long distance NaCl-stress sensing signaling molecule in seedling cotyledons. Exogenous melatonin (15µM)±NaCl (120mM) inhibit seedling growth, which is also correlated with NO availability, accumulation of potential superoxide anion (O 2 •- ) and peroxynitrite anion (ONOO - ), extent of tyrosine-nitration of proteins, spatial localization and activity of superoxide dismutase (SOD) isoforms. NO acts as a positive modulator of melatonin accumulation in seedling cotyledons as a long-distance signaling response. Modulation of superoxide anion and peroxynitrite anion content by melatonin highlights its crucial role in combating deleterious effects of ROS and reactive nitrogen species (RNS). Present findings provide evidence for an interaction between melatonin and NO in their effect on seedling growth under salt stress accompanying differential modulation of two SOD isoforms, i.e. Cu/Zn SOD and Mn SOD., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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.)

Nitric oxide (NO) acts in a concentration and redox-dependent manner to counteract oxidative stress either by directly acting as an antioxidant through scavenging reactive oxygen species (ROS), such as superoxide anions (O(2)(-)*), to form peroxynitrite (ONOO(-)) or by acting as a signaling molecule, thereby altering gene expression. NO can interact with different metal centres in proteins, such as heme-iron, zinc-sulfur clusters, iron-sulfur clusters, and copper, resulting in the formation of a stable metal-nitrosyl complex or production of varied biochemical signals, which ultimately leads to modification of protein structure/function. The thiols (ferrous iron-thiol complex and nitrosothiols) are also involved in the metabolism and mobilization of NO. Thiols bind to NO and transport it to the site of action whereas nitrosothiols release NO after intercellular diffusion and uptake into the target cells. S-nitrosoglutathione (GSNO) also has the ability to transnitrosylate proteins. It is an NO˙ reservoir and a long-distance signaling molecule. Tyrosine nitration of proteins has been suggested as a biomarker of nitrosative stress as it can lead to either activation or inhibition of target proteins. The exact molecular mechanism(s) by which exogenous and endogenously generated NO (or reactive nitrogen species) modulate the induction of various genes affecting redox homeostasis, are being extensively investigated currently by various research groups. Present review provides an in-depth analysis of the mechanisms by which NO interacts with and modulates the activity of various ROS scavenging enzymes, particularly accompanying ROS generation in plants in response to varied abiotic stress.

Detection of nitric oxide (NO) in plant cells is mostly undertaken using diaminofluorescein (DAF) dyes. Serious drawbacks and limitations have been identified in methods using DAF as a probe for NO detection. The present work reporting an alternative fluorescent probe for NO detection is thus proposed for varied applications in plant systems for physiological investigations. This method involves a simple, two-step synthesis, characterization, and application of MNIP-Cu {Copper derivative of [4-methoxy-2-(1H-napthol[2,3-d]imidazol-2-yl)phenol]} for specific and rapid binding with NO, leading to its detection in plant cells by epifluorescence microscopy and confocal laser scanning microscopy (CLSM). Using sunflower (Helianthus annuus L.) whole seedlings, hypocotyl segments, stigmas from capitulum, protoplasts, and isolated oil bodies, present investigations demonstrate the versatile nature of MNIP-Cu in applications for NO localization studies. MNIP-Cu can detect NO in vivo without any time lag (ex. 330-385 nm; em. 420-500 nm). It exhibits fluorescence both under anoxic and oxygen-rich conditions. This probe is specific to NO, which enhances its fluorescence due to MNIP-Cu complexing with NO and treatment with PTIO leads to quenching of fluorescence. It is relatively nontoxic when used at a concentration of up to 50 μM.

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.)

Strigolactones (SLs) play significant role in shaping root architecture whereby auxin-SL crosstalk has been observed in SL-mediated responses of primary root elongation, lateral root formation and adventitious root (AR) initiation. Whereas GR24 (a synthetic strigolactone) inhibits LR and AR formation, the effect of SL biosynthesis inhibitor (fluridone) is just the opposite (root proliferation). Naphthylphthalamic acid (NPA) leads to LR proliferation but completely inhibits AR development. The diffusive distribution of PIN1 in the provascular cells in the differentiating zone of the roots in response to GR24, fluridone or NPA treatments further indicates the involvement of localized auxin accumulation in LR development responses. Inhibition of LR formation by GR24 treatment coincides with inhibition of ACC synthase activity. Profuse LR development by fluridone and NPA treatments correlates with enhanced [Ca(2+)]cyt in the apical region and differentiating zones of LR, indicating a critical role of [Ca(2+)] in LR development in response to the coordinated action of auxins, ethylene and SLs. Significant enhancement of carotenoid cleavage dioxygenase (CCD) activity (enzyme responsible for SL biosynthesis) in tissue homogenates in presence of cPTIO (NO scavenger) indicates the role of endogenous NO as a negative modulator of CCD activity. Differences in the spatial distribution of NO in the primary and lateral roots further highlight the involvement of NO in SL-modulated root morphogenesis in sunflower seedlings. Present work provides new report on the negative modulation of SL biosynthesis through modulation of CCD activity by endogenous nitric oxide during SL-modulated LR development.

Dark-grown sunflower (Helianthus annuus L.) seedlings exhibit modulation of total superoxide dismutase (SOD;EC 1.15.1.1) activity in roots and cotyledons (10,000g supernatant) in response to salt stress (NaCl; 120 mM) through a differential, zymographically detectable, whole tissue activity of FeSOD and Cu/ZnSOD. Confocal laser scanning microscopic imaging (CLSM) has further shown that NaCl stress significantly influences differential spatial distribution of Cu/ZnSOD and MnSOD isoforms in an inverse manner. Dual action of nitric oxide (NO) is evident in its crosstalk with FeSOD and Cu/ZnSOD in seedling roots and cotyledons in control and NaCl(-) stress conditions. Cu/ZnSOD activity in the roots of 2 d old NaCl(-) stressed seedlings is enhanced in the presence of 125-1000 µM of NO donor (sodium nitroprusside; SNP) indicating salt sensitivity of the enzyme activity. Quenching of endogenous NO by cPTIO treatment (500, 1000 µM) lowers FeSOD activity in roots (-NaCl). Cotyledons from control seedlings show an upregulation of FeSOD activity with increasing availability of SNP (125-1000 µM) in the Hoagland irrigation medium. Quenching of NO by cPTIO provides evidence for an inverse correlation between NO availability and FeSOD activity in seedling cotyledons irrespective of NaCl stress. Variable response due to NO on SOD isoforms in sunflower seedlings reflects its concentration-dependent biphasic (pro- and antioxidant) nature of action. Differential induction of SOD isoforms by NO indicates separate intracellular signaling pathways (associated with their respective functional separation) operative in seedling roots as an early salt stress mechanism and in cotyledons as an early long-distance NaCl stress sensing mechanism.

Present investigations report the presence of strigolactones (SLs) and photomodulation of their biosynthesis in sunflower seedlings (roots, cotyledons and first pair of leaves) during early phase of seedling development. Qualitative analyses and characterization by HPLC, ESI-MS and FT-IR revealed the presence of more than one type of SLs. Orobanchyl acetate was detected both in roots and leaves. Five-deoxystrigol, sorgolactone and orobanchol were exclusively detected in seedling roots. Sorgomol was detectable only in leaves. HPLC eluted fraction from seedling roots and leaves co-chromatographing with GR24 (a synthetic SL) could also bring about germination in Orobanche cernua (a weed) seeds, which are established to exhibit SL - mediated germination, thereby indicating the SL identity of the eluates using this bioassay. SLs accumulation was always more in the roots of light-grown seedlings, it being maximum at 4 d stage. Although significant activity of carotenoid cleavage dioxygenase (CCD, the enzyme critical for SL biosynthesis) was detected in 2 d old seedling roots, SLs remained undetectable in cotyledons at all stages of development and also in the roots of 2 d old light and dark-grown seedlings. Roots of light-grown seedlings showed maximum CCD activity during early (2 d) stage of development, thereby confirming photomodulation of enzyme activity. These observations indicate the migration of a probable light-sensitized signaling molecule (yet to be identified) or a SL precursor from light exposed aerial parts to the seedling roots maintained in dark. Thus, a photomodulation and migration of SL precursor/s is evident from the present work.

A noteworthy metabolic signature accompanying oil body (OB) biogenesis during oilseed development is associated with the modulation of the oil body membranes proteins. Present work focuses on 2-dimensional polyacrylamide gel electrophoresis (2-D PAGE)-based analysis of the temporal changes in the OB membrane proteins analyzed by LC-MS/MS accompanying the onset of desiccation (20-30 d after anthesis; DAA) in the developing seeds of sunflower (Helianthus annuus L.). Protein spots unique to 20-30 DAA stages were picked up from 2-D gels for identification and the identified proteins were categorized into 7 functional classes. These include proteins involved in energy metabolism, reactive oxygen scavenging, proteolysis and protein turnover, signaling, oleosin and oil body biogenesis-associated proteins, desiccation and cytoskeleton. At 30 DAA stage, exclusive expressions of enzymes belonging to energy metabolism, desiccation and cytoskeleton were evident which indicated an increase in the metabolic and enzymatic activity in the cells at this stage of seed development (seed filling). Increased expression of cruciferina-like protein and dehydrin at 30 DAA stage marks the onset of desiccation. The data has been analyzed and discussed to highlight desiccation stage-associated metabolic events during oilseed development.

Understanding the physiological and biochemical basis of abiotic stress tolerance in plants has always been one of the major aspects of research aiming to enhance plant productivity in arid and semi-arid cultivated lands all over the world. Growth of stress-tolerant transgenic crops and associated agricultural benefits through increased productivity, and related ethical issues, are also the major concerns of current research in various laboratories. Interesting data on the regulation of abiotic stress tolerance in plants by serotonin and melatonin has accumulated in the recent past. These two indoleamines possess antioxidative and growth-inducing properties, thus proving beneficial for stress acclimatization. Present review shall focus on the modes of serotonin and melatonin-induced regulation of abiotic stress tolerance in plants. Complex molecular interactions of serotonin and auxin-responsive genes have suggested their antagonistic nature. Data from genomic and metabolomic analyses of melatonin-induced abiotic stress signaling have lead to an understanding of the regulation of stress tolerance through the modulation of transcription factors, enzymes and various signaling molecules. Melatonin, nitric oxide (NO) and calmodulin interactions have provided new avenues for research on the molecular aspects of stress physiology in plants. Investigations on the characterization of receptors associated with serotonin and melatonin responses, are yet to be undertaken in plants. Patenting of biotechnological inventions pertaining to serotonin and melatonin formulations (through soil application or foliar spray) are expected to be some of the possible ways to regulate abiotic stress tolerance in plants. The present review, thus, summarizes the regulatory roles of serotonin and melatonin in modulating the signaling events accompanying abiotic stress in plants.

Indoleamines regulate a variety of physiological functions during the growth, morphogenesis and stress-induced responses in plants. Present investigations report the effect of NaCl stress on endogenous serotonin and melatonin accumulation and their differential spatial distribution in sunflower (Helianthus annuus) seedling roots and cotyledons using HPLC and immunohistochemical techniques, respectively. Exogenous serotonin and melatonin treatments lead to variable effect on hypocotyl elongation and root growth under NaCl stress. NaCl stress for 48 h increases endogenous serotonin and melatonin content in roots and cotyledons, thus indicating their involvement in salt-induced long distance signaling from roots to cotyledons. Salt stress-induced accumulation of serotonin and melatonin exhibits differential distribution in the vascular bundles and cortex in the differentiating zones of the primary roots, suggesting their compartmentalization in the growing region of roots. Serotonin and melatonin accumulation in oil body rich cells of salt-treated seedling cotyledons correlates with longer retention of oil bodies in the cotyledons. Present investigations indicate the possible role of serotonin and melatonin in regulating root growth during salt stress in sunflower. Effect of exogenous serotonin and melatonin treatments (15 μM) on sunflower seedlings grown in the absence or presence of 120 mM NaCl substantiates their role on seedling growth. Auxin and serotonin biosynthesis are coupled to the common precursor tryptophan. Salt stress-induced root growth inhibition, thus pertains to partial impairment of auxin functions caused by increased serotonin biosynthesis. In seedling cotyledons, NaCl stress modulates the activity of N-acetylserotonin O-methyltransferase (HIOMT; EC 2.1.1.4), the enzyme responsible for melatonin biosynthesis from N-acetylserotonin., (© 2014 Scandinavian Plant Physiology Society.)

Seedlings exposed to salt stress are expected to show modulation of intracellular accumulation of sodium ions through a variety of mechanisms. Using a new methodology, this work demonstrates ouabain (OU)-sensitive ATPase activity in the roots of sunflower seedlings subjected to salt stress (120 mM NaCl). 9-Anthroylouabain (a derivative of ouabain known to inhibit Na(+), K(+) -ATPase activity in animal systems, EC 3.6.3.9) has been used as a probe to analyze OU-sensitive ATPase activity in sunflower (Helianthus annuus) seedling roots by spectrofluorometric estimation and localization of its spatial distribution using confocal laser scanning microscopy. Salt stress for 48 h leads to a significant induction of OU-sensitive ATPase activity in the meristematic region of the seedling roots. Calcium ions (10 mM) significantly inhibit enzyme activity and a parallel accumulation of sodium ions in the cytosol of the columella cells, epidermis and in the cells of the meristematic region of the roots is evident. As a rapid response to NaCl stress, the activity of OU-sensitive ATPase gets localized in the nuclear membrane of root protoplasts and it gets inhibited after treatment with calcium ions. Nuclear membrane localization of the OU-sensitive ATPase activity highlights a possible mechanism to efflux sodium ions from the nucleus. Thus, a correlation between OU-sensitive ATPase activity, its modulation by calcium ions and accumulation of sodium ions in various regions of the seedling roots, has been demonstrated using a novel approach in a plant system., (© 2013 Scandinavian Plant Physiology Society.)

Seed development in sunflower involves a gradual dehydration and accumulation of oil bodies in the cells of developing cotyledons during transition from 30 to 40 DAA stage. Reactive oxygen species (ROS) content decreased with seed maturation. NO content and NO contributed by putative nitric oxide synthase, however, did not change markedly. Superoxide dismutase (SOD) activity exhibited a peak at 30 DAA stage, indicating its scavenging role at the mid-stage of seed development. H₂O₂ produced as a result of SOD action is subsequently scavenged primarily by elevation of GR activity. Significant temporal differences were evident in GR and POD activity during seed development. Protein kinase C (PKC) activity also showed modulation during early stages of embryo and seed development. Use of PKC-specific fluorescent probe, Fim-1, and PKC inhibitors (staurosporine and bisindoylmaleamide) provided evidence for increase in PKC activity at 40 DAA stage with an increase in protein concentration (50 to 200 µg). Endogenous calcium content also increased with seed maturation. Tissue homogenates from 40 DAA stage showed enhanced fluorescence due to Fim-1-PKC binding in presence of calcium ions and its lowering due to calcium chelating agent (BAPTA). Western blot analysis revealed an increase in the intensity of 2 bands representing PKC with the advancement of seed maturation and their further upregulation by calcium. Present findings, thus, provide new information on the biochemical regulation of seed development in sunflower, with evidence for a possible correlation between calcium, ROS, their scavenging enzymes and "conventional" PKC activity.

Sunflower seedlings subjected to 120 mM NaCl stress exhibit high total peroxidase activity, differential expression of its isoforms and accumulation of lipid hydroperoxides. This coincides with high specific activity of phospholipid hydroperoxide glutathione peroxidase (PHGPX) in the 10,000g supernatant from the homogenates of 2-6 d old seedling cotyledons. An upregulation of PHGPX activity by NaCl is evident from Western blot analysis. Confocal laser scanning microscopic (CLSM) analysis of sections of cotyledons incubated with anti-GPX4 (PHGPX) antibody highlights an enhanced cytosolic accumulation of PHGPX, particularly around the secretory canals. Present work, thus, highlights sensing of NaCl stress in sunflower seedlings in relation with lipid hydroperoxide accumulation and its scavenging through an upregulation of PHGPX activity in the cotyledons.

Using NO specific probe (MNIP-Cu), rapid nitric oxide (NO) accumulation as a response to auxin (IAA) treatment has been observed in the protoplasts from the hypocotyls of sunflower seedlings (Helianthus annuus L.). Incubation of protoplasts in presence of NPA (auxin efflux blocker) and PTIO (NO scavenger) leads to significant reduction in NO accumulation, indicating that NO signals represent an early signaling event during auxin-induced response. A surge in NO production has also been demonstrated in whole hypocotyl explants showing adventitious root (AR) development. Evidence of tyrosine nitration of cytosolic proteins as a consequence of NO accumulation has been provided by western blot analysis and immunolocalization in the sections of AR producing hypocotyl segments. Most abundant anti-nitrotyrosine labeling is evident in proteins ranging from 25-80 kDa. Tyrosine nitration of a particular protein (25 kDa) is completely absent in presence of NPA (which suppresses AR formation). Similar lack of tyrosine nitration of this protein is also evident in other conditions which do not allow AR differentiation. Immunofluorescent localization experiments have revealed that non-inductive treatments (such as PTIO) for AR develpoment from hypocotyl segments coincide with symplastic and apoplastic localization of tyrosine nitrated proteins in the xylem elements, in contrast with negligible (and mainly apoplastic) nitration of proteins in the interfascicular cells and phloem elements. Application of NPA does not affect tyrosine nitration of proteins even in the presence of an external source of NO (SNP). Tyrosine nitrated proteins are abundant around the nuclei in the actively dividing cells of the root primordium. Thus, NO-modulated rapid response to IAA treatment through differential distribution of tyrosine nitrated proteins is evident as an inherent aspect of the AR development.

Lipoxygenase (linoleate:oxygen oxidoreductase; EC 1.13.11.12; LOX) catalyzes oxygenation of polyenoic fatty acids, which precedes the degradation of storage lipids during seed germination in sunflower. In the present work, it has been confirmed that 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA) produces fluorescence in presence of lipid hydroperoxides (LOX reaction products). This work provides new information on spatial localization of transiently enhanced LOX activity in protoplasts from 5 d old seedling cotyledons of sunflower (Helianthus annuus L. cv. Morden) by exploiting H(2)DCFDA as a probe for fluorescence detection from LOX activity sites. Use of LOX inhibitors [nordihydroguaiaretic acid (NDGA) and propyl gallate (PG)] confirms oil bodies as LOX activity sites. Oil body surface has been shown to possess LOX activity in 5 d old seedling cotyledons., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

Present work highlights the involvement of endogenous nitric oxide (NO) in sodium chloride (NaCl)-induced biochemical regulation of seedling growth in sunflower (Helianthus annuus L., cv. Morden). The growth response is dependent on NaCl concentration to which seedlings are exposed, they being tolerant to 40 mM NaCl and showing a reduction in extension growth at 120 mM NaCl. NaCl sensitivity of sunflower seedlings accompanies a fourfold increase in Na(+) /K(+) ratio in roots (as compared to that in cotyledons) and rapid transport of Na(+) to the cotyledons, thereby enhancing Na(+) /K(+) ratio in cotyledons as well. A transient increase in endogenous NO content, primarily contributed by putative NOS activity in roots of 4-day-old seedlings subjected to NaCl stress and the relative reduction in Na(+) /K(+) ratio after 4 days, indicates that NO regulates Na(+) accumulation, probably by affecting the associated transporter proteins. Root tips exhibit an early and transient enhanced expression of 4,5-diaminofluorescein diacetate (DAF-2DA) positive NO signal in the presence of 120 mM NaCl. Oil bodies from 2-day-old seedling cotyledons exhibit enhanced localization of NO signal in response to 120 mM NaCl treatment, coinciding with a greater retention of the principal oil body membrane proteins, i.e. oleosins. Abolition of DAF positive fluorescence by the application of specific NO scavenger [2-phenyl-4,4,5,5-tetramethyllimidazoline-1-oxyl-3-oxide (PTIO)] authenticates the presence of endogenous NO. These novel findings provide evidence for a possible protective role of NO during proteolytic degradation of oleosins prior to/accompanying lipolysis., (Copyright © Physiologia Plantarum 2010.)

Present work on indole-3-acetic acid (IAA)-induced adventitious rooting in sunflower hypocotyl highlights a clear demarcation of nitric oxide (NO)-dependent and NO-independent roles of auxin in this developmental process. Of the three phases of adventitious rooting, induction is strictly auxin-dependent though initiation and extension are regulated by an interaction of IAA with NO. A vital role of auxin-efflux transporters (PIN) is also evident from 1-napthylphthalamic acid (NPA)-triggered suppression of adventitious roots (AR). Use of actin depolymerizing agent, Latrunculin B (Lat B), has demonstrated the necessity of functional actin filaments in auxin-induced AR response, possibly through its effect on actin-mediated recycling of auxin transporter proteins. Thus, evidence for a linkage between IAA, NO and actin during AR formation has been established., (© 2010 Landes Bioscience)

Spatial distribution and compositional analyses of the lipidic constituents in pollen and stigma of sunflower (Helianthus annuus L. cv. Morden) were conducted using ultrastructural, histochemical, and biochemical analysis. Detection of secretions at the base of stigmatic papillae and neutral lipid accumulations on the surface of stigmatic papillae and between adjacent pseudopapillae demonstrates the semidry nature of stigma surface in sunflower. Pollen coat is richer in lipids (8%) than stigma (2.2%) on fresh weight basis. Nile Red-fluorescing neutral lipids are preferentially localized in the pollen coat. Neutral esters and triacylglycerols (TAGs) are the major lipidic constituents in pollen grains and stigma, respectively. Lignoceric acid (24:0) and cis-11-eicosenoic acid (20:1) are specifically expressed only in the pollen coat. Similar long-chain fatty acids have earlier been demonstrated to play a significant role during the initial signaling mechanism leading to hydration of pollen grains on the stigma surface. Lipase (EC 3.1.1.3) activity is expressed both in pollen grains and stigma. Stigma exhibits a better expression of acyl-ester hydrolase (EC 3.1.1.1) activity than that of observed in both the pollen fractions. Expression of two acyl-ester hydrolases (41 and 38 kDa) has been found to be specific to pollen coat. Specific expression of lignoceric acid (24:0) in pollen coat and localization of lipase in pollen and stigma have been discussed to assign possible roles that they might play during pollen-stigma interaction.

Oil bodies obtained from oilseeds have been exploited for a variety of applications in biotechnology in the recent past. These applications are based on their non-coalescing nature, ease of extraction and presence of unique membrane proteins-oleosins. In suspension, oil bodies exist as separate entities and, hence, they can serve as emulsifying agent for a wide variety of products, ranging from vaccines, food, cosmetics and personal care products. Oil bodies have found significant uses in the production and purification of recombinant proteins with specific applications. The desired protein can be targeted to oil bodies in oilseeds by affinity tag or by fusing it directly to the N or C terminal of oleosins. Upon targeting, the hydrophobic domain of oleosin embeds into the TAG matrix of oil body, whereas the protein fused with N and/or C termini is exposed on the oil body surface, where it acquires correct confirmation spontaneously. Oil bodies with the attached foreign protein can be separated easily from other cellular components. They can be used directly or the protein can be cleaved from the fusion. The desired protein can be a pharmaceutically important polypeptide (e.g. hirudin, insulin and epidermal growth factor), a neutraceutical polypeptide (somatotropin), a commercially important enzyme (e.g. xylanase), a protein important for improvement of crops (e.g. chitinase) or a multimeric protein. These applications can further be widened as oil bodies can also be made artificially and oleosin gene can be expressed in bacterial systems. Thus, a protein fused to oleosin can be expressed in Escherichia coli and after cell lysis it can be incorporated into artificial oil bodies, thereby facilitating the extraction and purification of the desired protein. Artificial oil bodies can also be used for encapsulation of probiotics. The manipulation of oleosin gene for the expression of polyoleosins has further expanded the arena of the applications of oil bodies in biotechnology., ((c) 2009 Elsevier Inc. All rights reserved.)

Oil bodies are spherical entities containing a triacylglycerol (TAG) matrix encased by a phospholipid monolayer, which is stabilized by oil body-specific proteins, principally oleosins. Biochemical investigations in the recent past have also demonstrated the expression of calcium-binding proteins, called caleosins, as a component of oil body membranes during seed germination. Using DM-Bodipy-phenylalkylamine (PAA; a fluorescent derivative of phenylalkylamine)-a fluorescent probe known to bind L-type calcium channel proteins, present investigations provide the first report on the localization and preferential accumulation of putative calcium channel proteins on/around oil bodies during peak lipolytic phase in protoplasts derived from dark-grown sunflower (Helianthus annuus L. cv Morden) seedling cotyledons. Specificity of DM-Bodipy-PAA labeling was confirmed by using bepridil, a non-fluorescent competitor of PAA while non-specific dye accumulation has been ruled out by using Bodipy-FL as control. Co-localization of fluorescence from DM-Bodipy-PAA binding sites (ex: 504 nm; em: 511 nm) and nile red fluorescing oil bodies (ex: 552 nm; em: 636 nm) has been undertaken by epifluorescence and confocal laser scanning microscopy (CLSM). It revealed the affinity of PAA-sensitive ion channels for the oil body surface. Findings from the current investigations highlight the significance of calcium and calcium channel proteins during oil body mobilization in sunflower.

Prior to and/or accompanying lipolytic degradation of triacylglycerols (TAGs) during seed germination in oilseeds, certain enzymatic and non-enzymatic signaling molecules are expressed on the oil body membranes. These include certain proteases, lipoxygenase, phospholipase A(2) and lipase. Although enough biochemical investigations have demonstrated their activities, recent developments in the in situ localization of these signaling molecules in germinating oilseeds, have enhanced our understanding in this field. This is evident from the temporal and spatial changes observed in the expression pattern of some of these molecules. Present review aims at providing an up-to-date account of these recent developments in the author's and other laboratories, which are largely based on fluorescence microscopic and confocal laser scanning microscopic (CLSM) imaging of the molecular changes using specific fluorescent probes. A model for the molecular events associated with oil body mobilization is also being presented.

The activity of a 65 kDa, cytosolic protease from sunflower seedling cotyledons coincides with the degradation of oleosins during seed germination. Further investigations carried out in this laboratory have demonstrated the probable association of a thiol-protease with oil bodies, leading to gradual degradation of oleosins during seedling growth. Evidence to this effect have been brought out through zymographic detection of protease activity from oil bodies, degradation of oleosins by electrophoretically eluted protease from the seedling cotyledons and inhibition of protease activity by thiol-protease inhibitor, such as N-ethylmaleimide (NEM). In addition to these biochemical evidence, visualization of thiol-protease activity has also been achieved by a novel fluorescence microscopic method and confocal imaging. It involves the uptake and binding of a fluorogenic thiol-protease inhibitor (fluorescein mercuric acetate, FMA) at the intracellular thiol-protease activity sites in protoplasts, leading to fluorescence emission at 523 nm following excitation at 499 nm. Maximum protease activity is observed in 4-d-old seedling cotyledons, coinciding with the phase of active triacylglycerol (TAGs) hydrolysis. All these observations provide evidence for the expression of the said thiol-protease activity on the oil body surface, leading to gradual proteolysis of oleosins during seed germination.

Auxin (indole-3-acetic acid) regulates caulonema differentiation as a result of gradual transitional events in the chloronema tip cells in moss protonema. This auxin action in the moss Funaria hygrometrica involves a rapid influx of calcium ions from the extracellular medium. This investigation demonstrates spatial and temporal changes in calmodulin (CaM) activation (formation of Ca(2+)-CaM complex) in the chloronema tip cells subjected to auxin treatment. Photomicroscopic localisation of the fluorescence (excitation at 365 nm and emission of 397 nm) from the tricomplex of Ca(2+)-CaM with trifluoperazine (TFP, a blocker of Ca(2+)-CaM action) shows a tip to base (tip high) gradient of Ca(2+)-CaM in the chloronema tip cells. Comparison of Ca(2+)-CaM-TFP fluorescence over time in the chloronema tip cells of wild type Funaria with the response in an auxin overproducer mutant (86.1) and an auxin deficient mutant (87.13) reveals the involvement of auxin in calmodulin activation as a rapid response prior to cell differentiation.