Sunflower Seedling Cotyledons Research Articles

Strigolactone and Auxin Applications on Cotyledon Senescence in Sunflower Seedlings under Salt Stress

Objective: Senescence is a programmed cell death process and is important in the growth, development and flowering process of the plant. Delaying senescence has a very important effect on agriculture in terms of product yield. Indole-3- acetic acid (IAA) and strigolactone (GR24) are growth regulators that affect plant development and senescence. Salt stress accelerates the senescence process. The aim of this study is to increase crop yield by delaying senescence by the application of auxin and GR24 under stress conditions, because the delay of senescence causes the prolongation of the vegetative process and the formation of more apical tips. In this case, the seedling produces more fruit. Materials and Methods: In this study, senescent cotyledons of sunflower seedlings were used as experimental material. Half of the developing sunflower seedlings were irrigated with Hoagland solution, and the other half was irrigated with 150 mM sodium chloride (NaCl) solution. IAA and GR24 were applied by spraying on seedlings that are grown both in Hoagland solution and under salt stress. Results: The degree of senescence of the cotyledons of the plants was determined in terms of the percentage of green area. When the green area percentage of cotyledons of seedlings grown in Hoagland solution was 50, all cotyledons were harvested. After that, fresh weight, pigment contents, total protein, malondialdehyde, and proline levels, peroxidase enzyme activities of cotyledons were determined. The application of IAA and GR24 to the cotyledons of seedlings grown in the salt medium significantly delayed the senescence. Conclusion: This study was conducted in the plant growth chamber under controlled conditions. Results showed that the application of IAA and GR24 to leaves can ameliorate the adverse effects of salt stress and delay senescence due to the activation of chlorophyll components and modulation of photosynthesis as well as antioxidant defense capacity. The effect of IAA is more precise when all analyzes are considered. More importantly, showed that all findings (except MDA and Proline) IAA and GR24 promote senescence in Hoagland in this research. Delaying senescence contributes to basic science. It is desired to increase fruit and vegetable yield by delaying senescence. It is suggested that this information can be used practically in the field of agriculture. On the other hand in this study, we can say that IAA and relatively GR24 can play an important role in the protection of plants in agricultural areas in salt stress.

Melatonin–Nitric Oxide Interaction Modulates Catalase Activity and Hydrogen Peroxide Homeostasis in Sunflower Seedling Cotyledons Accompanying NaCl Stress

Melatonin–Nitric Oxide Interaction Modulates Catalase Activity and Hydrogen Peroxide Homeostasis in Sunflower Seedling Cotyledons Accompanying NaCl Stress

Nitric oxide modulates polyamine homeostasis in sunflower seedling cotyledons under salt stress

ABSTRACTFree 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.

Nitric oxide and light co-regulate glycine betaine homeostasis in sunflower seedling cotyledons by modulating betaine aldehyde dehydrogenase transcript levels and activity

ABSTRACTGlycine 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.

Tyrosine nitration of cytosolic peroxidase is probably triggered as a long distance signaling response in sunflower seedling cotyledons subjected to salt stress.

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.

Melatonin and nitric oxide modulate glutathione content and glutathione reductase activity in sunflower seedling cotyledons accompanying salt stress

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.

Nitric oxide and iron modulate heme oxygenase activity as a long distance signaling response to salt stress in sunflower seedling cotyledons

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.

Photomodulation of fatty acid composition and lipoxygenase activity in sunflower seedling cotyledons

The dynamics of fatty acid composition is an important metabolic index of seed germination and seedling development process. Present investigations indicate a preferential mobilization of fatty acids, particularly linoleic acid, out of the oil bodies in sunflower seedling cotyledons. A lack of selectivity in the utilization of saturated fatty acids during seed germination is also evident. Lipoxygenase (LOX) may play a role in the reconstitution of membrane lipid composition as cells expand during seed germination. LOX activity is absent in sunflower seeds and appears during germination. Photo modulation of LOX activity is evident by its enhanced expression in light-grown seedling cotyledons. Since sunflower LOX activity is easily solubilized by mild detergent, such as 2 % Triton X-100, the association of LOX with the membrane does not seem to be very strong unlike other membrane proteins, such as oleosins, which can be extracted only by stringent procedures. Sunflower LOX has only one pH optimum with linoleic and linolenic acids as substrates, indicating the presence of single isoform in the total soluble protein (TSP) and oil body (OB) extracts. OB-associated LOX exhibits substrate-dependent variation in its pH optima. Specific activity of TSP and OB-associated LOX increases as seedling growth progresses. Sunflower TSP-LOX activity is inhibited by some hydrophobic thiols. TSP as well as OB-associated LOX activity in sunflower cotyledons is modulated by Ca2+ availability in a negative way. A critical role of LOX is, thus, evident during seed germination in sunflower.

Signaling role of phospholipid hydroperoxide glutathione peroxidase (PHGPX) accompanying sensing of NaCl stress in etiolated sunflower seedling cotyledons

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.

Co-localization of putative calcium channels (phenylalkylamine-binding sites) on oil bodies in protoplasts from dark-grown sunflower seedling cotyledons

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.

Evidence for the probable oil body association of a thiol-protease, leading to oleosin degradation in sunflower seedling cotyledons

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.

Spatial and temporal changes in lipase activity sites during oil body mobilization in protoplasts from sunflower seedling cotyledons

Hydrolysis of triacylglycerols (TAGs) catalyzed by lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) action, is the principal biochemical event during oil body mobilization in germinating oilseeds. Employing a fluorescence microscopic technique developed in the author’s laboratory, a shift in the intracellular lipase activity has been demonstrated in the protoplasts of sunflower seedling cotyledons during seed germination. Lipase activity is primarily confined to protein storage vacuoles (PSVs) in 1 d old seedling cotyledons. At 2 d old stage, a relocalization of lipase activity begins and activity can be observed both on PSVs and oil bodies. At later stages of development (3–6 d), smaller PSVs coalesce into a large vegetative vacuole devoid of lipase activity. During this phase, lipase activity is confined to oil bodies only and maximum activity is detected in 4 d old seedlings, coinciding with maximum rate of lipolysis. Thus, present investigations on protoplasts from seedling cotyledons provide evidence for intracellular shift in lipase activity to sites of TAG hydrolysis (oil bodies) and also show a structural and functional reorganization of PSVs.

Lepidimoide Promotes Light-Induced Chlorophyll Accumulation in Cotyledons of Sunflower Seedlings.

The effect of disaccharide lepidimoide on light-induced chlorophyll accumulation was studied in cotyledons of sunflower (Helianthus annuus L.) seedlings and detached cucumber (Cucumis sativus L.) cotyledons. From studies on the structure-activity relationships of lepidimoide, its analogs, and sucrose with respect to light-induced chlorophyll accumulation in the cotyledons of sunflower seedlings, both lepidimoide and the free carboxylic acid of lepidimoide (lepidimoic acid) showed the highest promoting activity, whereas the hydrogenated lepidimoide, which lacks a double bond in the C4, 5 position in uronic acid, showed lower activity than lepidimoide; however, sucrose exhibited very weak activity. These results suggest that lepidimoide acts as a new type of plant growth regulator, not simply as a carbon source providing energy. Lepidimoide promoted not only light-induced chlorophyll accumulation in sunflower cotyledons but also light-induced 5-aminolevulinic acid content, which is considered to be a rate-limiting step in chlorophyll biosynthesis. Lepidimoide with cytokinin stimulated the accumulation of chlorophyll and 5-aminolevulinic acid additively. In detached cucumber cotyledons, lepidimoide also promoted light-induced chlorophyll accumulation. These results indicate that lepidimoide, in cooperation with cytokinin, causes light-induced chlorophyll accumulation in the cotyledons of several dicot plant species by affecting the level of 5-aminolevulinic acid. Key Words. 5-Aminolevulinic acid accumulation-Chlorophyll accumulation-Cucumis sativus-Cytokinin-Helianthus annuus-Lepidimoic acid-Lepidimoide-Structure-activity relationship

Turnover of Catalase Heme and Apoprotein Moieties in Cotyledons of Sunflower Seedlings

The turnover of catalase apoprotein and catalase heme was studied in cotyledons of sunflower (Helianthus annuus L.) seedlings by density labeling of apoprotein and radioactive labeling of heme moieties. The heavy isotope (50% (2)H(2)O) and the radioactive isotope ([(14)C]5-aminolevulinic acid) were applied either during growth in the dark (day 0-2.5) or in the light (day 2.5 and 5). Following isopycnic centrifugation of catalase purified from cotyledons of 5-day-old seedlings, superimposition curve fitting was used to determine the amounts of radioactive heme moieties in native and density-labeled catalase. Data from these determinations indicated that turnover of catalase heme and apoprotein essentially was coordinate. Only small amounts of heme groups were recycled into newly synthesized apoprotein during growth in the light, and no evidence was found for an exchange of heme groups in apoprotein moieties. It followed from these observations that degradation of catalase apoprotein was slightly faster than that of catalase heme. A degradation constant for catalase apoprotein of 0.263 per day was determined from the data on heme recycling and the degradation constant of catalase heme determined previously to be 0.205 per day (R Eising, B Gerhardt [1987] Plant Physiol 84: 225-232).

Effects of the embryonic axis and exogenous growth regulators on sunflower cotyledon storage protein mobilization

Cotyledons of sunflower seedlings (Helianthus annuus L. cv. Giant gray stripe) expand and their protein content first rises then begins to decrease during the first three days of growth. Storage protein structures, which are visible with scanning electron microscopy, undergo modification that leads to storage protein disappearance by day 4 post‐imbibition. Expansion of cotyledons detached from seeds prior to imbibition is greatly reduced, total protein levels remain high, and storage protein structures remain visible in cells of these cotyledons. Incubation of excised cotyledons in 1.0 μM benzyladenine or kinetin increases the rates of cotyledon expansion and storage protein loss to levels higher than in intact seedling cotyledons, Incubation in 10 μM indole‐3‐acetic acid inhibits cotyledon expansion and protein mobilization. More rapid hydrolysis of storage proteins in cotyledons of intact seedlings or detached cotyledons treated with cytokinin is further indicated in day 2 specimens by SDS‐polyacrylamide gel electrophoresis. These results suggest a possible mechanism for regulation of cotyledon development by interactions of the promotive effects of cytokinin and inhibitory effects of auxin.

Reinigung und Eigenschaften der Phenoloxidase aus Sonnenblumen

Phenoloxidase was isolated from cotyledons of sunflower seedlings by precipitation with acetone, with ammonium sulfate, and by Sephadex chromatography. The purified enzyme, as judged by substrate specificity, is an o -diphenol oxidase. Neither monophenols, nor meta - or para -diphenols, were exidized. The enzyme was inhibited by chelating agents and by sulfhydryl compounds in the sequence KCN > Na-DDTC > 2-mercaptoethanol > cystine-HCl > thiourea > sodium azide. Although the phenoloxidase was not inhibited by 2-mercaptobenzthiazole, this compound did cause a distinct lag-phase in enzyme activity. Four isoenzymes with similar mobility but differnt activity were separated by disk electrophoresis. Similar differences were observed in bands obtained by isoelectric focusing. The isoelectric points were found at p H 5.3, 5.8, 6.0 and 6.4. The absorption spectrum of the purified enzyme was typical for proteins and without marked specialities. Aus den Keimblättern der Sonnenblume (Helianthus annuus) wurde Phenoloxidase isoliert und durch Aceton- bzw. Ammoniumsulfatfällung und Chromatographie an Sephadex weitgehend gereinigt. In seiner Substratspezifität erwies sich das reine Enzym als o-Diphenoloxidase. Monophenole und ebenso Meta- und Paradiphenole wurden nicht angegriffen. Komplexbildner und Sulfhydryl-Verbindungen hemmten die Enzymaktivität mit abnehmender Wirkung in folgender Reihenfolge: KCN > NA-DDTC > 2-Mercaptoäthanol > Cystein · HCl > Thioharnstoff > Natriumazid. 2-Mercaptobenzthiazol hemmte nicht, verursachte aber eine ausgeprägte Lag-Phase. Durch die Disk-Elektrophorese wurden 4 Isoenzyme identifiziert, deren Banden nahe beeinander lagen und unterschiedliche Aktivität zeigten. Etwa die gleichen Aktivitätsunterschiede wurden auch bei den Banden beobachtet, die bei der isoelektrischen Fokussierung der Enzyrnlösung erhalten wurden. Die isoelektrischen Punkte der Isoenzyme lagen bei p H 5,3, 5,8, 6,0 und 6,4. Die gereinigte Phenoloxidase hatte das für Proteine typische Absorptionsspektrum ohne ausgeprägte Besonderheiten.