Estimation of substances with the cytokinin activity in studies on the correlation between the cotyledon and its axillary bud in pea (Pisum sativum L.)

The content of substances showing the cytokinin activity was estimated on decapitated and one-cotyledon-deprived pea plants during that period when the promoting effect of the cotyledon excision had not yet been manifested. Results of the bioassay showed that after the excision an increase in the level of substances with cytokinin activity occurred only in cotylars growing in axillas of these excised cotyledons. These results coincide with earlier data about the content of gibberellins and auxins in the same object.     

Changes in Paramagnetic Manganese (Mn2+) and Related Enzyme Activities in Isolated Cucumber Cotyledons During Growth: II. EFFECT OF 6-FURFURYLAMINOPURINE

Changes in the activities of IAA oxidase, peroxidase, ascorbicacid utilization (AAU), and in the level of paramagnetic manganese(Mn²⁺) have been studied during kinetin-induced growth of theisolated cucumber cotyledons in light or in dark. In kinetin-treatedcotyledons exposed to light, inhibition in the level of paramagneticmanganese corresponds with an enhancement in IAA oxidase activity.The level of paramagnetic manganese shows an inverse correlationwith IAA oxidase activity. In darkness the level of Mn²⁺ doesnot show the same correlation with IAA oxidase activity as inthe light. Kinetin stimulates peroxidase activity both in thelight and in darkness. Enhancement of IAA oxidase activity andno corresponding change in the level of paramagnetic manganeseindicates that the oxidation of IAA in dark-grown, kinetin-treatedcotyledons is brought about by peroxidase. It appears that thephenolic cofactors required for the oxidation of manganese andIAA may be limiting in kinetin-treated cotyledons in darkness.Thus in the light, IAA oxidation seems to be brought about byperoxidase as well as manganese, whereas in darkness it is mediatedby peroxidase alone. Increase in IAA oxidase activity duringkinetin-induced growth of the isolated cotyledons is incompatiblewith the idea that increased IAA oxidase activity would limitthe availability of auxin for growth. Kinetin does not mimicthe action of light on IAA oxidase activity; on the contrary,it removes the inhibitory effect of light on IAA oxidase activityprobably through the synthesis of IAA oxidase activators.     

Differences in the activity and distribution of peroxidases from three different portions of germinating Brassica oleracea seeds

Peroxidase (POD, EC 1.11.1.7) activity, cellular localization and isozyme patterns were investigated in the seed integument, cotyledon and embryo axis of Brassica oleracea cv. Cappuccio during pregermination and seedling growth. Seeds started to germinate after 24 h of imbibition. POD activity was localized in the pigmented layer of the integument and in procambial strands of the cotyledon and embryo axis in the first 24 h of imbibition. It was localized in the integumental cells of palisade, pigmented and aleurone layers and in epidermal, meristematic, procambial cells and xylem elements of the root and hypocotyl after 48 h of imbibition. POD activity increased during germination and early seedling growth: in the integument, it reached a maximum value after 72 h of imbibition, in the embryo axis and cotyledons, it increased up to 144 h of imbibition. The increase in peroxidase activity was accompanied by the appearance of new isozymes correlated with the development of seedling tissues. The isozyme profile was characterized by nine peroxidases: isoperoxidase of 50 kDa peculiar to integuments, that of 150 kDa to cotyledons and that of 82 kDa to the embryo axis. During pregerminative phase isozymes of 84 kDa were detected in the integument and cotyledons, of 48.5 kDa in the embryo axis. After germination, peroxidase activity and the complexity of the isozyme pattern increased, suggesting that they play a relevant role after rupture of the integument.     

Sequential release of both basic and acidic isoperoxidases to the media of suspension cultured cells of Capsicum annuum

Summary The establishment of suspension cell cultures from trimmed cotyledons of pepper (Capsicum annuum L.) provides a new experimental system for studying the relationship between release of peroxidase (EC 1.11.1.7) into the free intercellular spaces and plant cell growth. In contrast with several other species, the total peroxidase activity in the medium increased continuously during the post-exponential growth phase of the pepper cell culture, and this was correlated with the growth inhibition of pepper cells cultivated in suspension. The increase in the peroxidase activity in the culture medium was the consequence of a differential release of isoperoxidases, prominently marked by a primary release of basic isoperoxidases, followed by a strong increase in the level of acidic isoperoxidases. Thus, pepper cells cultures constitute a new experimental system for studying the regulation of the sequential release of basic and acidic isoperoxidases, which occurs during the growth cessation of plant cells.     

Salicylic acid mediated by the oxidative burst is a key molecule in local and systemic responses of cotton challenged by an avirulent race of Xanthomonas campestris pv malvacearum

We analyzed the production of reactive oxygen species, the accumulation of salicylic acid (SA), and peroxidase activity during the incompatible interaction between cotyledons of the cotton (Gossypium hirsutum) cv Reba B50/Xanthomonas campestris pv malvacearum (Xcm) race 18. SA was detected in petioles of cotyledons 6 h after infection and 24 h post inoculation in cotyledons and untreated leaves. The first peak of SA occurred 3 h after generation of superoxide (O(2)(.-)), and was inhibited by infiltration of catalase. Peroxidase activity and accumulation of SA increased in petioles of cotyledons and leaves following H(2)O(2) infiltration of cotyledons from 0.85 to 1 mM. Infiltration of 2 mM SA increased peroxidase activity in treated cotyledons and in the first leaves, but most of the infiltrated SA was rapidly conjugated within the cotyledons. When increasing concentrations of SA were infiltrated 2. 5 h post inoculation at the beginning of the oxidative burst, the activity of the apoplastic cationic O(2)(.-)-generating peroxidase decreased in a dose-dependent manner. We have shown that during the cotton hypersensitive response to Xcm, H(2)O(2) is required for local and systemic accumulation of SA, which may locally control the generation of O(2)(.-). Detaching cotyledons at intervals after inoculation demonstrated that the signal leading to systemic accumulation of SA was emitted around 3 h post inoculation, and was associated with the oxidative burst. SA produced 6 h post infection at HR sites was not the primary mobile signal diffusing systemically from infected cotyledons.     

Enzymatic changes in gourd and bean cotyledons during ageing and the effect of detopping

Cotyledons of gourd (Cucurbita maxima Duchesne) and bean (Phaseolus vulgaris L.) were used to study the changes in the activities of catalase, peroxidase, acid inorganic pyrophosphatase and alkaline inorganic pyrophosphatase during ageing and the diversion in such changes that occur when cotyledon senescence was retarded by detopping the seedlings above the cotyledons. Catalase, acid inorganic pyrophosphatase and alkaline inorganic pyrophosphatase activities declined during the senescence of the cotyledons. When cotyledon senescence was retarded by detopping as marked by the increase in the levels of chlorophyll and protein, there was also an increase in the activities of these enzymes. Peroxidase activity, on the other hand, increased during the senescence of the cotyledons and detopping the seedlings resulted in a further increase in the peroxidase activity. It can be suggested that some root factor(s) probably cytokinin(s) is (are) mobilised into the cotyledons of the detopped seedlings which otherwise would have been mobilised into the shoot apices, and help retard or even reverse the senescence of the cotyledons.     

Regulation of ascorbate peroxidase activity in dark-grown radish cotyledons by a catalase inhibitor, 3-Amino-1,2,4-Triazole

The present study was performed to see the physiological role of cytosolic ascorbate peroxidase (APX) and its relationship to other enzymes involved in the H₂O₂ scavenging metabolism, and also to elucidate the regulation of APX expression in dark-grown radish (Raphanus sativus L. cv Taiwang) cotyledons. To do so, 3-amino-l,2,4-triazole (aminotriazole), a known specific inhibitor of catalase, was used to simulate a catalase-deficient phenomenon in cotyledons. Aminotriazole, in very low concetration (10^-4 M), inhibited remarkably the development of catalase activity in cotyledons during dark germination. This inhibition of catalase by aminotriazole, however, did not result in any significant changes in the growth response and the H₂O₂ level of developing cotyledons. In addition, the development of guaiacol peroxidase (GPX) activity was also not significantly affected. Unlike GPX, cytosolic APX activity was induced rapidly and reached a 1.7-fold increase in aminotriazole treated cotyledons at day 7 after germination. However,in vitro incubation of cytosolic APX preparation from cotyledons with aminotriazole did not result in any significant change in activity. One cytosolic APX isozyme (APXa) band involved in this APX activation was predominantly intensified in a native polyacrylamide gel by activity staining assay. This means that this APXa isozyme seems to play a key role in the expression of cytosolic APX activity. On the other hand, 2-day-old control seedlings treated with exogenous 1 mM H₂O₂ for 1 h showed a significant increase of cytosolic APX acitivity even in the absence of aminotriazole. Also, 2 μM cycloheximide treatment substantially inhibited the increase of APX activity due to aminotriazole. Based on these results, we suggest that a radish cytosolic APX could probably be substituted for catalase in H₂O₂ removal and that the expression of APX seems to be regulated by a change of endogenous H₂O₂ level which couples to APX protein synthesis in a translation stage in cotyledons.     

Changes in IAA, tryptophan and activity of soluble peroxidase associated with zygotic embryogenesis in Araucaria angustifolia (Brazilian pine)

In A. angustifolia seeds the highest values of freeIAA occurred in the embryonic axis, at the initial phases of development. Thesevalues decreased sharply coincident with the increase of IAA with thedifferentiation of cotyledons and seed elongation. During seed development,tryptophan concentrations varied inversely with free IAA and directly withconjugated IAA. An increase in peroxidase activity was followed by a decreaseinfree IAA in the embryo axis, and in conjugated IAA in the megagametophyte.Megagametophyte tissues did not exhibit significant variation in free IAAduringseed development. Following the stage where cotyledons arise, tryptophandecreased in the megagametophyte and increased in the cotyledons and embryonicaxis.     

Oxidation of an organosulfur xenobiotic by microsomes from soybean cotyledons

Methiocarb, an aromatic-alkyl sulfide insecticide was enzymatically oxidized into its sulfoxide by microsomes from soybean cotyledons. No further oxidation into sulfone was detected. Distribution of the sulfoxidase activity was studied in soybean seedlings and found maximal in cotyledons. Subcellular fractionation of cotyledons homogenates indicated that the activity was almost entirely associated with the microsomal fraction. Sulfoxidation of methiocarb did not require cofactors such as NAD(P)H. Nevertheless, the sulfoxidase did not act as a peroxidase.     

Lipid peroxidation and peroxide-scavenging enzymes associated with accelerated aging of peanut seed

Accelerated aging is known to reduce seed viability and vigor in many crop species. The phenomenon is due in part to aging-induced lipid peroxidation, which has the potential to damage membranes of the seed tissues. This study was undertaken to evaluate the effect of accelerated aging on germinability and several physiological characteristics related to peroxidation in the seed of two peanut cultivars. Accelerated aging was achieved by incubating seed at 45°C and 79% relative humidity in a closed chamber for 3, 6, or 9 days. The results indicate that accelerated aging inhibited seed germination and seedling growth. Enhanced lipid peroxidation and increased peroxide accumulation were observed in the axis and cotyledons of aged seed. Accelerated aging also inhibited the activity of superoxide dismutase, peroxidase, ascorbate peroxidase, and lipoxygenase. Seed axes appeared to be more susceptible to aging than cotyledons. The changes in germination and physiological activities, expressed as a function of aging duration, were similar in the two cultivars, despite differences in their seed weight.     

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.     

A COMPARATIVE INVESTIGATION OF PEROXIDASES FROM GERMINATING PEANUTS (ARACHIS HYPOGAEA): ELECTROPHORESIS

Dormant seed and organs of 0-, 1-, 2-, 5-, 8-, 11-, and 14-day-old plants of Arachis hypogaea L. were homogenized in phosphate buffer and the lipid-free extracts analyzed for benzidine and pyrogallol peroxidases using starch-gel electrophoresis. On a wet weight basis, one weak band of benzidine peroxidase activity was detected in dormant cotyledons and three bands in 1-day cotyledons. In 5-day tissue, activity had increased significantly; at 14 days, the number of bands had decreased but staining intensity was maintained. In the extract from dormant axis, a single cathodic site of benzidine peroxidase activity was observed; however, on day two there was a marked increase in the number of bands and intensity of reaction in epicotyl and hypocotylradicle tissues. By day 14, the number and density of bands had decreased noticeably in the epicotyl and hypocotyl. Extracts from 14-day roots exhibited more sites of reaction and greater intensity of staining of benzidine peroxidase than at five days of growth. Localized areas of activity at Rf -0.44 and -0.52 were present in extracts of all four organs when either benzidine or pyrogallol was used as the hydrogen donor. Although marked similarity existed between banding patterns of organs, qualitative and quantitative ontogenetic differences in peroxidases were apparent.     

Effects of ageing on peroxidase activity and localization in radish (Raphanus sativus L.) seeds

Peroxidase activity was assayed in crude extracts of integument, cotyledons and embryo axis of radish seeds, deteriorated under accelerated ageing conditions. Over five days of ageing, in which germination decreased from 100 to 52%, the enzyme activity in integument was higher than that in other seed parts, increasing in the first days of ageing and then decreasing sharply in extremely aged seeds. Polyacrylamide gel electrophoresis analysis showed four peroxidase isoenzymes with MM of 98, 52.5, 32.8 and 29.5 kDa in the embryo axis of unaged seeds, and only the 32.8 and 29.5 kDa MM isoforms in the integument and cotyledons. In these parts of the seed, only the 29.5 kDa MM isoenzyme increased in activity in early days of ageing and decreased there-after. In the embryo axis, the 29.5 kDa MM isoenzyme activity increased slowly in the first day of ageing, while the 98 and 52.5 kDa MM isoenzyme activities disappeared. A cytochemical localization of peroxidase activity in the various tissues showed that main differences between unaged and extremely aged seeds occurred in the embryo axis.     

Inhibitory effect of polyamines on the activity of endopeptidase in mung bean cotyledons

The activity of cysteine endopeptidase (EP) in the cotyledons of mung bean seeds increased with time after germination. When cotyledons were excised from the embryonic axis in the course of seedling growth, the activity of EP in the excised cotyledon markedly dropped during the following incubation of 1 d. However, the level of EP protein in excised cotyledons, as examined by immunoblotting, was similar to that in axis-attached cotyledons at the corresponding stage. Thus, it seems that the low activity of EP in excised cotyledons is not due to a decrease in the content of EP protein, but due to a loss of the activity of existing EP. Treatment of attached cotyledons with polyamines (PAs; putrescine and spermidine [Spd]) resulted in a decrease in EP activity, while the same PA-treatment brought about little alteration in the level of EP protein. This indicates that PAs somehow produce an inhibitory effect on the activity of EP. Axis-removal resulted in an accumulation of Spd in the cotyledon. The possibility is suggested that PA, especially Spd, is involved in the inhibition of EP activity in excised mung bean cotyledons.     

Changes in antioxidative enzymes in cucumber cotyledons during natural senescence: comparison with those during dark-induced senescence

Changes in 7 antioxidative enzymes in naturally senescent cotyledons of cucumber ( Cucumis sativus ) were investigated. The activities of superoxide dismutase (SOD; EC 1.15.1.1), catalase (EC 1.11.1.6), dehydroascorbate reductase (EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2) gradually decreased during the progression of senescence, while those of ascorbate peroxidase (APX; EC 1.11.1.11) and guaiacol peroxidase (GPX; EC 1.11.1.7) gradually increased. The activity of monodehydroascorbate reductase (MDAR; EC 1.6.5.4) was not significantly changed. Western blot analysis showed that the protein level of mitochondrial SOD gradually declined. The protein level of catalase transiently decreased and then increased in the later stages of senescence, despite the decrease in its activity. The overall behavior was markedly different from that found in cotyledons of artificially senescing seedlings transferred into darkness; the activities of SOD, catalase, APX, GPX and GR gradually increased.     

Biochemical changes in the germinating seeds ofTrigonella foenum graecum L. in relation tos-triazine herbicides

Thes-triazine herbicides studied (atrazine and simazine) inhibited the growth of the seedlings ofTrigonella foenum graecum L.(Viciaceae). These herbicides caused a decrease in the contents of total water soluble carbohydrates and reducing sugars. Pentose sugars increased in the cotyledons. The total protein and free amino acid contents were enhanced. The activity of peroxidase was inhibited in the cotyledons, but was enhanced in the seedling axes.     

Salicylic acid and ethylene pathways are differentially activated in melon cotyledons by active or heat-denatured cellulase from Trichoderma longibrachiatum

Infiltration of cellulase (EC 3.2.1.4) from Trichoderma longibrachiatum into melon (Cucumis melo) cotyledons induced several key defense mechanisms and hypersensitive reaction-like symptoms. An oxidative burst was observed 3 hours after treatment and was followed by activation of ethylene and salicylic acid (SA) signaling pathways leading to marked induction of peroxidase and chitinase activities. The treatment of cotyledons by heat-denatured cellulase also led to some induction of peroxidase and chitinase activities, but the oxidative burst and SA production were not observed. Co-infiltration of aminoethoxyvinil-glycine (an ethylene inhibitor) with the active cellulase did not affect the high increase of peroxidase and chitinase activities. In contrast, co-infiltration of aminoethoxyvinil-glycine with the denatured enzyme blocked peroxidase and chitinase activities. Our data suggest that the SA pathway (induced by the cellulase activity) and ethylene pathway (induced by heat-denatured and active protein) together coordinate the activation of defense mechanisms. We found a partial interaction between both signaling pathways since SA caused an inhibition of the ethylene production and a decrease in peroxidase activity when co-infiltrated with denatured cellulase. Treatments with active or denatured cellulase caused a reduction in powdery mildew (Sphaerotheca fuliginea) disease.     

Proteome, salicylic acid, and jasmonic acid changes in cucumber plants inoculated with Trichoderma asperellum strain T34

Trichoderma spp. is one of the most commonly used biological control agents against plant pathogens. This fungus produces changes in plant metabolism, thus increasing growth and enhancing resistance to biotic and abiotic stresses. However, its modes of action remain to be defined. In the first hours of interaction between cucumber plant roots and Trichoderma asperellum strain T34, salicylic and jasmonic acid levels and typical antipathogenic peroxidase activity increase in the cotyledons to different degrees depending on the applied concentration of the fungi. The use of 2-DE protein profiling and MS analysis allowed us to identify 28 proteins whose expression was affected in cotyledons after cucumber root colonization by Trichoderma applied at high concentrations: 17 were found to be up-regulated while 11 were down-regulated. Proteins involved in ROS scavenging, stress response, isoprenoid and ethylene biosynthesis, and in photosynthesis, photorespiration, and carbohydrate metabolism were differentially regulated by Trichoderma. The proteome changes found in this study help to give an understanding of how Trichoderma-treated plants become more resistant to pathogen attacks through the changes in expression of a set of defence-oriented proteins which can directly protect the plant or switch the metabolism to a defensive, nonassimilatory state.     

Desiccation-induced changes in lipid peroxidation, superoxide level and antioxidant enzymes activity in neem (Azadirachta indica A. Juss) seeds

The freshly harvested mature neem seeds (42.2 % seed moisture content) with 100 % viability deteriorate when naturally desiccated to below 10.9 %. The desiccation-induced loss of viability was closely associated with over accumulation of superoxide anion and lipid peroxidation products both in the embryonic axes and cotyledons. The levels of superoxide anion and lipid peroxidation products were higher in axes compared to cotyledons. Superoxide dismutase activity was not much affected, both in the axes and cotyledons of 100 % viable seeds during desiccation from 42.2 % to 10.9 % seed moisture content. Steep rise in its activity was observed during drying below lowest safe moisture content (LSMC). Activities of catalase and peroxidase exhibited substantially higher levels in the 100 % viable seeds dehydrated up to LSMC. Their activities declined sharply in seeds with water content below LSMC. Impairment of catalase and peroxidase activities possibly lead to enhanced accumulation of reactive oxygen species. The accumulation of superoxide anion, lipid peroxidation and differential expression of superoxide dismutase and catalse/peroxidase activities in response to desiccation (below LSMC) is discussed to explain the intermediate storage physiology of neem seeds.